AU2024412280A1 - Engineered bacterium, construction method therefor and use thereof - Google Patents

Abstract

An engineered bacterium for improving tryptophan yield, a construction method therefor, and a use thereof. A strain capable of tolerating a high concentration of tryptophan was obtained by screening. Genome sequencing and protein sequence analysis of the strain revealed that some proteins in the strain had undergone point mutations, and these mutants can improve the tryptophan yield. In order to further improve the tryptophan yield, modifications are made to a protein sequence expressed by a fadR gene or a protein sequence expressed by a pepD gene in a starting bacterium. Such modifications enable the obtained engineered bacterium to have a higher tryptophan yield than the starting bacterium. In the case of large-scale production, in a 5 L fermentation tank, the tryptophan yield can reach 62.38±5.80 g/L, and the sugar-to-acid conversion rate can reach 24.1%. Compared with the original bacterium, the tryptophan yield is increased by 1.48 folds, and the sugar-to-acid conversion rate is increased by 1.26 folds. The biological material and the use thereof relate to the technical field of molecular biology and have wide practical application value.

Description

ENGINEERED STRAIN AND ENGINEERED STRAIN AND THE THE CONSTRUCTION CONSTRUCTIONMETHOD METHOD AND AND USE USE THEREOF THEREOF CROSS CROSS REFERENCE TORELATED REFERENCE TO RELATEDAPPLICATIONS APPLICATIONS

[0001] Thisapplication

[0001] This applicationclaims claimspriority priorityto to Chinese ChinesePatent PatentApplication ApplicationNo. No.202311807065.2, 202311807065.2, filed filed on on December 26,2023, December 26, 2023,the theentire entire contents contents of of which are incorporated which are incorporated herein herein by by reference. reference.

TECHNICALFIELD TECHNICAL FIELD

[0002]

[0002] TheThe present present disclosure disclosure relatesrelates to the to the technical technical field offield of molecular molecular biology, biology, and in and in particular, to an engineered strain, a construction method, and a use thereof. particular, to an engineered strain, a construction method, and a use thereof.

BACKGROUND BACKGROUND

[0003] L-tryptophan,

[0003] L-tryptophan, as an as an essential essential amino amino acid, is acid, widelyisused widely used across in fields in fields the across the food, feed, food, feed,

and pharmaceuticalindustries. and pharmaceutical industries. TheThe biosynthesis biosynthesis of of tryptophan tryptophan primarily primarily involves involves three three

modules:the modules: the central central carbon metabolism(CCM) carbon metabolism (CCM) pathway, pathway, the shikimate the shikimate (SHIK) (SHIK) pathway, pathway, and and the the chorismate (CHA) chorismate (CHA) pathway. pathway. Due Due to itstolong its long biosynthetic biosynthetic route, route, requirement requirement for multiple for multiple

precursors, and strong feedback inhibition, the production efficiency of tryptophan is relatively precursors, and strong feedback inhibition, the production efficiency of tryptophan is relatively

low, resultingininthe low, resulting theproduction production of industrial of industrial mature mature strains strains struggling struggling to meet to meetdemand. market market demand. Withthe With the continuous continuousadvancement advancementof of synthetic synthetic biology, biology, numerous numerous metabolic metabolic engineering engineering

strategies havebeen strategies have been applied applied to the to the construction construction offactories of cell cell factories for efficient for efficient production production of L- of L- tryptophan. Although tryptophan. Although rational rational modification modification of of metabolic metabolic pathways pathways is goal-oriented is goal-oriented and and often often

yields significant yields significantresults, results,microorganisms microorganisms are areinherently inherentlycomplex complex systems. Previous systems. Previous studies studies

showed thatthe showed that the expression expressionof of cytidine cytidine deaminase deaminaseonly onlyslightly slightly increases increases the the mutation rate and mutation rate and

knownmetabolic known metabolic networks networks or or engineering engineering strategies strategies cannot cannot achieve achieve thethe purpose purpose of of rapidly rapidly

enhancingproduction. enhancing production.

SUMMARY SUMMARY

[0004]

[0004] ToTo solve solve theabove the above problem, problem, a straincapable a strain capable ofof toleratinghigh-concentration tolerating high-concentrationtryptophan tryptophan is is obtained obtained through through tolerance-based screening. Genome tolerance-based screening. Genome sequencing sequencing and protein and protein sequence sequence

analysis on the strain reveal that certain proteins of the strain undergo point mutations, and these analysis on the strain reveal that certain proteins of the strain undergo point mutations, and these

mutantsis mutants is capable of enhancing capable of tryptophanproduction. enhancing tryptophan production.Through Through inducing inducing mutations mutations in thein the strain that are strain that are unrelated unrelatedtotothe themain main metabolic metabolic pathways pathways foramino for target targetacid amino acid and synthesis synthesis and glycolysis, aa strain glycolysis, strainwith withdesirable desirableproperties is obtained. properties is obtained.However, due to However, due to the the high high degree degree of of

complexityofofthe complexity the metabolic metabolicnetwork networkofofananorganism, organism,thetheobtained obtainedmutation mutation resultsexhibit results exhibit significant uncertainty significant uncertainty andand are are difficult difficult to accurately to accurately predict predict and regulate and regulate at the at thestage early earlyofstage of research. Those research. Those skilledininthe skilled theart art would nottypically would not typically consider consider whether whethermodifications modificationstotothese these proteins could proteins could be be beneficial beneficial for fortryptophan tryptophan production. Inspiredbybythe production. Inspired themutagenesis mutagenesis system system

based on based on the the fusion fusion of of cytidine cytidine deaminase andT7T7RNA deaminase and RNA polymerase, polymerase, it is it is found found that that fusing fusing cytidine deaminase cytidine withthe deaminase with the αsubunit subunitofofEscherichia Escherichiacoli coliRNA RNA polymerase polymerase enables enables accelerated accelerated mutagenesis to levels that support efficient adaptive evolution in Escherichia coli without mutagenesis to levels that support efficient adaptive evolution in Escherichia coli without compromising cellviability. compromising cell viability. Mutagenic Mutagenic evolution evolution is an is an efficientindustrial efficient industrial approach approachfor forrapidly rapidly enhancing the production enhancing the productioncapacity capacityofofmicrobial microbialstrains. strains. When When combined combined with with advanced advanced techniques such techniques such as as genome genomesequencing, sequencing, it itenables enablesthe theintegration integrationof of productivity productivity improvement improvement with mechanistic investigation, providing a foundation and direction for subsequent rational with mechanistic investigation, providing a foundation and direction for subsequent rational engineering. At same engineering. At the the same time, time, it it boosts boosts the productivity the productivity of strain of andstrain and strengthens strengthens international international competitiveness. competitiveness.

[0005] First

[0005] First aspect aspect of the of the present present disclosure disclosure provides provides an engineered an engineered strain for strain for increasing increasing

tryptophan production, tryptophan production, and andthe the engineered engineeredstrain strain is is obtained obtained by by modification fromaa parent modification from parent strain. In some strain. In someembodiments, embodiments,thethe engineered engineered strain strain comprises comprises a gene a gene encoding encoding at least at least oneone of of a a

pepDmutant pepD mutantprotein proteinororaafadR fadRmutant mutantprotein. protein.A second A second aspect aspect of the of the present present disclosure disclosure

provides aa method provides forconstructing method for constructingan anengineered engineeredstrain strain with with increased increasedtryptophan tryptophanproduction. production. In In some embodiments, some embodiments, thethe method method comprises comprises obtaining obtaining the the engineered engineered strain strain by modifying by modifying at at least least one ofaapepD one of pepD protein protein or aor a fadR fadR protein protein of a parent of a parent strain;strain; anda under and under a same culture same culture

condition, a tryptophan production of the engineered strain is higher than a tryptophan condition, a tryptophan production of the engineered strain is higher than a tryptophan

production of the parent strain. production of the parent strain.

[0006]

[0006] A A thirdaspect third aspectofofthe thepresent present disclosure disclosure provides provides aa biomaterial, biomaterial, which is used which is for used for

increasing increasing tryptophan production. tryptophan production.

[0007]

[0007] InInsome some embodiments, embodiments, the the biomaterial biomaterial comprises comprises a mutant a mutant protein, protein, and mutant and the the mutant protein includes protein includes aa sequence havingat sequence having at least least 80% sequenceidentity 80% sequence identity to to an an amino aminoacid acidsequence sequence shown inSEQ shown in SEQID ID NO:NO: 1 or1 a orsequence a sequence having having at least at least 80%80% sequence sequence identity identity to amino to an an amino acid acid

sequence shown sequence shown inin SEQ SEQ ID ID NO: NO: 2. 2.

[0008]

[0008] InInsome some embodiments, embodiments, the the biomaterial biomaterial comprises comprises a DNAa molecule, DNA molecule, and theand DNAthe DNA

moleculecomprises molecule comprisesa agene geneencoding encoding thethe mutant mutant protein. protein.

[0009]

[0009] InInsome some embodiments, embodiments, the the biomaterial biomaterial comprises comprises a gene a gene expression expression cassette, cassette, and and the the

gene expressioncassette gene expression cassette includes includes the the mutant protein or mutant protein or the the gene gene encoding the mutant encoding the mutantprotein. protein.

[0010]

[0010] InInsome some embodiments, embodiments, the the biomaterial biomaterial comprises comprises a recombinant a recombinant vector, vector, and the and the

recombinantvector recombinant vectorincludes includesthe themutant mutantprotein proteinororthe the gene geneencoding encodingthe themutant mutantprotein. protein.

[0011]

[0011] A A fourthaspect fourth aspectofofthe thepresent presentdisclosure disclosureprovides providesaause useof of an an engineered engineeredstrain strain in in increasing increasing tryptophan production. tryptophan production.

[0012]

[0012] In In thethe present present disclosure, disclosure, the production the production of tryptophan of tryptophan specifically specifically refers refers to the to the ability ability

of of the the strain straintoto produce produceand andaccumulate tryptophan in accumulate tryptophan in aa culture culture medium. medium.

[0013]

[0013] AsAs used used herein,the herein, theterm term"protein" “protein”ofofthe thepresent presentdisclosure disclosurehas has the the meaning meaningcommonly commonly understood by those of ordinary skill in the art. understood by those of ordinary skill in the art.

2

[0014] The

[0014] The “pepD "pepD mutant mutant protein” protein" of the of the present present disclosure disclosure is is obtained obtained by by introducing introducing a a

mutation into mutation into an an amino aminoacid acidsequence sequenceshown shownin in SEQ SEQ ID NO: ID NO: 4. Specifically, 4. Specifically, the pepD the pepD mutantmutant

protein is obtained by substituting amino acid residues at positions 21, 225, and 484 of the amino protein is obtained by substituting amino acid residues at positions 21, 225, and 484 of the amino

acid sequence acid shownininSEQ sequence shown SEQID ID NO:NO: 4 with 4 with threonine, threonine, alanine, alanine, andand lysine, lysine, respectively.In respectively. In addition, the addition, the scope scope of of the thepresent presentdisclosure disclosurealso encompasses also encompasses enzymes derivedfrom enzymes derived from Escherichiacoli Escherichia coli that that share share more more than than 80%, preferably90%, 80%, preferably 90%,more more preferably preferably 95%, 95%, andand mostmost

preferably over preferably 99%homology over 99% homology with with the the amino amino acidacid sequence sequence shown shown in SEQinID SEQ NO: ID NO: 1 and 1 and possess the possess the function function of of cleaving cleaving dipeptides dipeptides with with an an unblocked N-terminus.SuchSuch unblocked N-terminus. enzymes enzymes are are also withinthe also within thescope scope of of thethe present present disclosure. disclosure.

[0015] Similarly,the

[0015] Similarly, the"fadR “fadRmutant mutant protein”ofofthe protein" thepresent presentdisclosure disclosureisis obtained obtained by by introducing introducing aa mutation into an mutation into an amino acid sequence amino acid sequenceshown shownin in SEQ SEQ ID NO: ID NO: 5. Specifically, 5. Specifically, the fadR the fadR mutant mutant

protein is obtained by substituting amino acid residues at positions 140 and 171 of the amino protein is obtained by substituting amino acid residues at positions 140 and 171 of the amino

acid acid sequence shownininSEQ sequence shown SEQID ID NO:NO: 5 with 5 with threonine threonine and and isoleucine, isoleucine, respectively. respectively.

Additionally, Additionally, the the scope scope of of the the present present disclosure disclosurealso alsoencompasses proteins derived encompasses proteins derived from from

Escherichiacoli Escherichia coli that that share share more than 80%, more than preferablymore 80%, preferably morethan than90%, 90%, more more preferably preferably over over

95%,and 95%, andmost mostpreferably preferablyabove above 99% 99% homology homology withamino with the the amino acid sequence acid sequence shown shown in in SEQ ID SEQ ID NO:22and NO: andpossess possessdual dualDNA-binding DNA-binding transcriptional transcriptional regulator regulator activity.SuchSuch activity. proteins proteins are are also also

within the scope of the present disclosure. within the scope of the present disclosure.

[0016] The

[0016] The term term “exogenous” "exogenous" of the of the present present disclosure disclosure refers refers to to a a system system containing containing a a

substance that is substance that isnot notoriginally originallypresent. present. For For example, if aa gene example, if gene encoding an enzyme encoding an enzymethat thatisis not not originally presentinina agiven originally present given strain strain is is introduced introduced into into that that strain strain by means by means such as such as transformation, transformation,

and the and the enzyme enzyme isissubsequently subsequentlyexpressed expressedininthe thestrain, strain, then then the the enzyme is considered enzyme is considered “exogenous” to that strain. "exogenous" to that strain.

[0017] The

[0017] The term term “enhancing” "enhancing" not not onlyonly refers refers to to an an increase increase in in thefunctional the functionaleffect effect beyond beyondthe the original leveldue original level duetotoananincrease increase in the in the intrinsic intrinsic activity activity of the of the protein protein itself, itself, but but maybealso be may also

achieved through achieved throughatat least least one one of of the the following following approaches: increasing the approaches: increasing the copy numberofofthe copy number the nucleotides encoding nucleotides encodingthe the protein; protein; modifying theregulatory modifying the regulatory sequence sequenceofofthe thegene geneencoding encodingthethe protein; replacing protein; replacing the theregulatory regulatorysequence sequence of of the the gene gene encoding the protein encoding the protein on on the the chromosome chromosome

with a strong active sequence; substituting the gene encoding the protein with a mutant gene to with a strong active sequence; substituting the gene encoding the protein with a mutant gene to

enhance the enhance the protein’s protein's activity; activity; or introducing or introducing modifications modifications into theinto genethe gene the encoding encoding protein the on protein on

the chromosome the chromosome to to enhance enhance protein protein activity,and activity, andmay may alsonon-limitedly also non-limitedly include include any any existing existing

methodthat method that can canenhance enhanceprotein proteinactivity activity or or enhance activity of enhance activity of the the introduced introduced protein protein compared compared

to its endogenous activity. to its endogenous activity.

[0018] The

[0018] The term term “activityofofintroduced "activity introducedprotein" protein”has hasthe theconventional conventionalmeaning meaning understood understood by by

those skilled those skilled in inthe theart, andand art, may maybe beimplemented using known implemented using knownmethods methods in in thethe field,including field, includingbut but

3 not limited not limited to: to:inserting insertinga polynucleotide a polynucleotidecomprising comprising aapolynucleotide polynucleotide sequence encodingthe sequence encoding the protein into protein into the thechromosome; and/orcloning chromosome; and/or cloningthe thepolynucleotide polynucleotideinto intoaavector vectorand andintroducing introducingitit into into aa microorganism; and/orincreasing microorganism; and/or increasing the the copy copynumber numberofof thepolynucleotide the polynucleotide directlyononthe directly the chromosome; and/or chromosome; and/or modifying modifying the the promoter promoter of the of the polynucleotide polynucleotide encoding encoding the protein the protein to to enhance transcription enhance transcription initiation initiation rate; rate; and/or and/or modifying modifying the transcription the transcription of the polynucleotide of the polynucleotide encoding the encoding the protein protein to enhance to enhance its activity; its activity; and/or and/or altering altering the translation the translation regulatory regulatory sequencessequences of the of the mRNA carrying mRNA carrying thethe polynucleotide polynucleotide encoding encoding the the protein protein to to enhance enhance translation translation intensity; intensity; and/or modifying the polynucleotide itself to improve mRNA stability, protein stability, or and/or modifying the polynucleotide itself to improve mRNA stability, protein stability, or relieve feedback relieve inhibition of feedback inhibition of the theprotein. It may protein. It also non-limitedly may also include any non-limitedly include knownmethod any known method capable capable ofofintroducing introducing protein protein activity. activity.

[0019] The

[0019] The term term “vector” "vector" referstotoa aDNA refers DNA construct construct comprising comprising a polynucleotide a polynucleotide sequence sequence

encoding a target encoding a target protein, protein, which which is operably is operably linkedlinked to suitable to suitable regulatory regulatory sequencessequences to enable the to enable the

expression expression ofof the the target target protein protein in ainhost a host cell. cell. Once introduced Once introduced into a host into a suitable suitable cell,host the cell, the

vector may replicate or function independently of the host genome, or it may be integrated into vector may replicate or function independently of the host genome, or it may be integrated into

the host the host genome. These genome. These vectors vectors maymay not not be specifically be specifically limited limited as as long long as as thevector the vectorisis replicable replicable in inthe thehost hostcell. cell. Examples of vectors Examples of vectors include include natural natural or or recombinant plasmids, recombinant plasmids,

cosmids, viruses, and cosmids, viruses, and bacteriophages. For bacteriophages. For example, example, pWE15, pWE15, pET, pET, pUC vectors, pUC vectors, or the or the like. like.

In addition,bybyinserting In addition, insertingthethe vector vector intointo the the chromosome chromosome ofcell, of a host a host thecell, the polynucleotide polynucleotide

encoding the endogenous encoding the endogenous targetprotein target proteinononthe thechromosome chromosomemay may be replaced be replaced with with a modified a modified

polynucleotide. Insertion polynucleotide. Insertionofofa apolynucleotide polynucleotideinto intothe thechromosome chromosomemay may be carried be carried out out using using

any methodknown any method knownin in thethe art,including art, includingbut butnot notlimited limited to to homologous recombination. homologous recombination. The The

polynucleotide includes polynucleotide includes DNA DNA andand RNARNA encoding encoding the target the target protein protein and be and may may be inserted inserted into into the the chromosome chromosome of of thethe hostcell host cellinin any anyform, form,asas long longas as it it can can be be expressed expressed in in the the host hostcell. cell. For For

example, the example, the polynucleotide polynucleotide may may be be introduced introduced into the into the host host cell cell in its in itsform native native form and/or and/or in the in the

form of an form of an expression expression cassette. cassette. AnAn expression expression cassetterefers cassette referstotoaa genetic genetic construct construct containing containing

all essential elements required for expression, and may also be a self-replicating expression all essential elements required for expression, and may also be a self-replicating expression

vector. ItIt may vector. mayinclude includea apromoter promoter operably operably linked linked to to thepolynucleotide, the polynucleotide,transcription transcription termination signals, termination signals, ribosome bindingdomains, ribosome binding domains,and andtranslation translationtermination terminationsignals. signals.

[0020] The

[0020] The term term “weakening” "weakening" of the of the present present disclosure disclosure refers refers to to reducing,weakening, reducing, weakening, diminishing, diminishing, or or eliminating eliminating the the activity activity of a of a protein, protein, such such as an as an enzyme. enzyme. In specificIn specific

embodiments, weakening embodiments, weakening of enzyme of enzyme activity activity may may be achieved be achieved by partially by partially or completely or completely

knocking out the gene encoding the enzyme, mutational inactivation or partial inactivation of the knocking out the gene encoding the enzyme, mutational inactivation or partial inactivation of the

gene, alteringthe gene, altering thegene gene promoter promoter or translation or translation regulatory regulatory region region to totranscription weaken weaken transcription or or translation, modifying translation, modifying the the gene gene sequence to reduce sequence to reducemRNA mRNA stability stability or or enzyme enzyme structural structural

stability, stability, regulating thegene regulating the geneviavia sRNA, sRNA, or combinations or combinations of these of these methods, methods, etc. etc.

4

[0021] The

[0021] The term term “host "host cell”ofofthe cell" thepresent presentdisclosure disclosurehas has the the meaning meaningcommonly commonly understood understood by by aa person person ofofordinary ordinary skill skill in in thethe art,i.e., art, i.e.,aastrain strainthat thatcontains contains a protein a protein or or a protein a protein mutant mutant

thereof. In other words, the present disclosure may utilize any host cell as long as the cell thereof. In other words, the present disclosure may utilize any host cell as long as the cell

contains contains the the target targetprotein proteinorora a mutant mutantthereof thereofand andisis capable ofofproducing capable producingtryptophan. Thehost tryptophan. The host cells cells may may bebe derived derived fromfrom Escherichia Escherichia coli coli (E. (E. Coli). Coli). Specifically, Specifically, the host ofthe thehost of the present present

disclosure referstotoa astrain disclosure refers straincapable capableof of producing producing tryptophan, tryptophan, i.e., i.e., in in a culture a culture medium,medium, the the bacteria can bacteria can produce andaccumulate produce and accumulatetryptophan tryptophan oror secretetryptophan secrete tryptophaninto intothe theculture culture medium, medium, thereby obtaining extracellular free tryptophan. In particular, it refers to a strain having the thereby obtaining extracellular free tryptophan. In particular, it refers to a strain having the

ability abilityto toaccumulate accumulate more tryptophancompared more tryptophan comparedto to thewild-type the wild-typeororparent parentstrain. strain. ToTo confer confer

the ability the abilitytotoproduce producetryptophan tryptophan to toaastrain, strain,conventional breeding conventional breedingmethods methods may be employed, may be employed, such ascultivating such as cultivatingauxotrophic auxotrophic mutants, mutants, analog-resistant analog-resistant strains,strains, or metabolic or metabolic control mutants control mutants

capable of producing capable of tryptophan,asas well producing tryptophan, well as as cultivating cultivating recombinant strains with recombinant strains with enhanced enhanced

activity activity of ofenzymes related to enzymes related to amino acid biosynthesis, amino acid biosynthesis, or or combinations of these combinations of these methods. methods.

[0022] The

[0022] The term term "a “a pepD pepD mutant mutant protein protein and/or and/or a fadR a fadR protein protein mutant” mutant" of the of the present present

disclosure hasa ameaning disclosure has meaning routinely routinely understood understood by thoseby of those of the skill in skillartinand themay artbeand may be implemented implemented byby methods methods known known in the in the art,art, including including butbut notnot limitedto,to,inserting limited inserting the the polynucleotide comprising polynucleotide comprisingpolynucleotide polynucleotidesequences sequences encoding encoding the the protein protein into into thethe chromosome, chromosome,

and/or cloning the and/or cloning the polynucleotide into aa vector polynucleotide into vector and and introducing introducing it itinto intomicroorganisms, microorganisms, and/or and/or

directly directly increasing increasing the thecopy copy number of the number of the polynucleotide onthe polynucleotide on the chromosome. chromosome. It may It may also also

non-limitedly include non-limitedly include any any known known methods methods capable capable of introducing of introducing protein protein activity. activity.

[0023] Those

[0023] Those skilledininthe skilled theart art understand understandthat that when whenmutating mutatinga awild-type wild-typepolypeptide polypeptide to to

enhance activity,identifying enhance activity, identifying the the sites sites thatthat achieve achieve the desired the desired effecteffect is of greater is of greater importance. importance.

Based on the teachings of the present disclosure, those skilled in the art would substitute serine at Based on the teachings of the present disclosure, those skilled in the art would substitute serine at

position 21, glycine at position 225, and alanine at position 484 of the amino acid sequence of position 21, glycine at position 225, and alanine at position 484 of the amino acid sequence of

the pepD protein with threonine, alanine, and lysine, respectively; and substitute alanine at the pepD protein with threonine, alanine, and lysine, respectively; and substitute alanine at

position 140 position and leucine 140 and leucine at at position position 171 171 of of the the amino amino acid acid sequence of the sequence of the fadR protein with fadR protein with threonine and isoleucine, followed by testing the relevant activities of these mutants. threonine and isoleucine, followed by testing the relevant activities of these mutants.

[0024]

[0024] In In addition, addition, it would it would not not be be difficult difficult for a for a person person of ordinary of ordinary skill in skill in to the art theknow art to know that that

changing a few changing a few amino amino acid acid residues residues in certain in certain regionsregions of a polypeptide, of a polypeptide, e.g., in non-significant e.g., in non-significant

regions, does not essentially alter the biological activity, e.g., sequences obtained by appropriate regions, does not essentially alter the biological activity, e.g., sequences obtained by appropriate

substitution ofcertain substitution of certainamino amino acids acids do affect do not not affect the activity the activity (see, (see, e.g., e.g., WatsonWatson et al, Molecular et al, Molecular

Biology of Biology of The The Gene, Gene,Fourth FourthEdition, Edition,1987. 1987.The The Benjamin/Cummings Benjamin/Cummings Pub. Pub. Co. Co. P224). P224). As As such, such, aa person personofofordinary ordinary skill skill in the in the art art would would be to be able able to carry carry outsubstitution out this this substitution and ensure and ensure

that the resulting molecule still has the desired biological activity. that the resulting molecule still has the desired biological activity.

[0025] Therefore,ititis

[0025] Therefore, is obvious obviousto to obtain obtain further further mutants of the mutants of the pepD and/or fadR pepD and/or fadRproteins proteinsof of the present disclosure and their mutants, which still retain the corresponding functions and the present disclosure and their mutants, which still retain the corresponding functions and activities, activities, through additional through additional mutagenesis. mutagenesis. For example, For example, it is wellitknown is well known to those to those skilled skilled in the in the art art that that adding adding orordeleting deleting several several amino amino acid acid residues residues at either at either terminus terminus of a polypeptide, of a polypeptide, preferably 1 to 20 residues, more preferably 1 to 15 residues, even more preferably 1 to 10 preferably 1 to 20 residues, more preferably 1 to 15 residues, even more preferably 1 to 10 residues, still more preferably 1 to 3 residues, and most preferably 1 residue, does not affect the residues, still more preferably 1 to 3 residues, and most preferably 1 residue, does not affect the function function ofofthe theresulting resulting mutant. mutant. Forofease For ease of purification, purification, the persons the skilled skilled often persons often attach a attach a

6×His tagtotoeither 6×His tag eitherterminus terminus of the of the obtained obtained protein, protein, anda such and such taggedaprotein taggedretains proteintheretains same the same

function function as as the the protein proteinwithout without the the6×His 6×His tag. Thepresent tag. The presentdisclosure disclosureshould shouldtherefore thereforeinclude include conserved mutantsobtained conserved mutants obtainedbased basedonon thepresent the presentdisclosure. disclosure.

[0026] Compared

[0026] Compared with with the theart, prior prior theart, the present present disclosure disclosure has the beneficial has the following followingeffects. beneficial effects.

[0027] The

[0027] The present present disclosureobtains disclosure obtainsthe theengineered engineeredstrain strainwith withananincreased increasedtryptophan tryptophan production compared production compared toto theparent the parentstrain strain by by modifying modifyinga aprotein proteinsequence sequenceexpressed expressed by by a fadR a fadR

gene or aa protein gene or protein sequence expressedby sequence expressed byaapepD pepDgene gene inin a agenome genomeof of thethe parent parent strain;the strain; theparent parent strain strain being being aa tryptophan tryptophan producing strain; modifying producing strain; the fadR modifying the geneis fadR gene is to to weaken its expression weaken its expression

level or its level or its protein activity, or protein activity, or even evenknock knock out out the the fadRfadR gene; gene; and modifying and modifying the pepD the pepD gene is to gene is to

mutate the pepD gene, enhance its expression level or its protein activity. In the case of large- mutate the pepD gene, enhance its expression level or its protein activity. In the case of large-

scale scale production, production, the the tryptophan tryptophan production reaches 62.38±5.80 production reaches 62.38±5.80g/L g/Lininaa55LLfermenter, fermenter,with withaa glucose-to-tryptophanyield glucose-to-tryptophan yield of of 24.1%. 24.1%.Compared Compared to original to the the original strain, strain, thethe tryptophan tryptophan

production is increased by 1.48-fold, and the glucose-to-tryptophan yield is increased by 1.26- production is increased by 1.48-fold, and the glucose-to-tryptophan yield is increased by 1.26-

fold. fold.

[0028] The

[0028] The deposit deposit information information of of thetheengineered engineered strainofofthe strain thepresent presentdisclosure disclosureis is provided provided

below. below.

[0029] Escherichia

[0029] Escherichia coliIBEWQ-624 coli IBEWQ-624 was deposited was deposited in theinChina the China CenterCenter for Culture for Type Type Culture Collection Collection(CCTCC) on August (CCTCC) on August 19, 19, 2024, 2024,with witha deposit number a deposit CCTCC number CCTCCNO: NO: M M 20241821, 20241821, and and

the deposit the deposit address: address: No. No. 299, 299, Bayi Road, Wuchang Bayi Road, Wuchang District,Wuhan District, Wuhan City, City, Hubei Hubei Province, Province, China. China.

BRIEF BRIEF DESCRIPTION DESCRIPTION OF OF THE THEDRAWINGS DRAWINGS

[0030] FIG.

[0030] FIG. 1 1 isisa aschematic schematicdiagram diagram illustratingtryptophan illustrating tryptophanfermentation fermentationlevels levelsofofaa strain strain IBEWQ-62 IBEWQ-62 and and strains strains derived derived from from the the strain strain IBEWQ-62 IBEWQ-62 with different with different weakening weakening degrees degrees of a of a fadR geneor/and fadR gene or/andwith withdifferent different enhancement enhancement degrees degrees of of a a pepD pepD gene gene in in a genome. a genome. The The

diagramindicates diagram indicates the the enhanced enhancedexpression expressionofofthe thepepD pepDgene gene significantlyimproves significantly improvesthethe

fermentation performancewhile fermentation performance whileweakening weakening or even or even completely completely knocking knocking outfadR out the the fadR gene gene

yields the yields the best bestimprovement in fermentation improvement in fermentationperformance. performance.

DETAILED DESCRIPTION DETAILED DESCRIPTION

6

[0031] One

[0031] One of of embodiments embodiments of the of the present present disclosure disclosure provides provides an engineered an engineered strain, strain, thethe

engineered strain engineered strain is is obtained obtained by modification by modification from a from parenta strain, parent and strain, and the engineered the engineered strain strain comprises comprises aa gene geneencoding encodingatatleast least one one of of aa pepD mutantprotein pepD mutant proteinororaafadR fadRmutant mutantprotein. protein.

[0032]

[0032] InInsome some embodiments, embodiments, the the pepDpepD mutant mutant protein protein includes includes at least at least one one of mutations of mutations S21T, S21T,

G225A, andA484K. G225A, and A484K. For example, For example, a mutation a mutation involved involved in the in themutant pepD pepD protein mutant protein may be may be

mutations S21T, mutations S21T,G225A, G225A,or or A484K, A484K, denoted denoted S21T S21T as pepD as pepD , pepDG225A , pepDG²²A, or ,pepD, or pepD A484K , respectively. respectively.

As another As another example, example,the themutation mutationinvolved involvedininthe thepepD pepD mutant mutant protein protein maymay be the be the mutations mutations

S21T and G225A, S21T and G225A,mutations mutations G225A G225Aand andA484K, A484K,orormutations mutations S21T S21Tand and A484K, A484K,denoted denotedas as S21T, G225A G225A, pepD G225A, G225A, A484K A484K or pepl A484K, A484K,S21 S21, respectively. pepD pepD S21T, , pepD or pepD , respectively.

[0033]

[0033] InInsome some embodiments, embodiments, the the mutation mutation involved involved in pepD in the the pepD mutant mutant protein protein may be may be

mutations S21T, mutations S21T,G225A, G225A,andand A484K, A484K, denoted denoted A484K,A484K, as pepD as pepD S21, S21, G225A G225A . S21T

[0034] The

[0034] The pepDrefers pepD²¹ refers to the to the serine serine at at position position 21 21 of of anan amino amino acid acid sequence sequence (SEQ(SEQ ID NO: ID NO:

4) of the pepD protein being substituted by threonine, and the relevant activity of this protein 4) of the pepD protein being substituted by threonine, and the relevant activity of this protein

mutant is then tested. mutant is then tested.

[0035]

[0035] The pepDG225A refers The pepDG²²A refers to to the the glycine glycineatatposition position 225 225 of the of the amino amino acid sequence acid sequence of the of the

pepD protein being substituted by alanine, and the relevant activity of this protein mutant is then pepD protein being substituted by alanine, and the relevant activity of this protein mutant is then

tested. tested.

A484Kto the alanine at position 484 of the amino acid sequence of the

[0036] The

[0036] The pepD pepD refers refers to the alanine at position 484 of the amino acid sequence of the pepD protein being substituted by lysine, and the relevant activity of this protein mutant is then pepD protein being substituted by lysine, and the relevant activity of this protein mutant is then

tested. tested.

[0037]

[0037] InInsome some embodiments, embodiments, the the pepDpepD mutant mutant protein protein includes includes a sequence a sequence havinghaving at least at least 80% 80%

sequence identity to sequence identity to an an amino acid sequence amino acid sequenceshown shownin in SEQ SEQ ID NO: ID NO: 1. In1.some In embodiments, some embodiments, the pepD the mutantprotein pepD mutant proteinincludes includesaasequence sequencehaving havingatatleast least 85% 85%sequence sequence identitytotothe identity theamino amino acid acid sequence shownininSEQ sequence shown SEQID ID NO:NO: 1. some 1. In In some embodiments, embodiments, the pepDthe pepDprotein mutant mutant protein includes includes a a sequence havingatat least sequence having least 90% sequenceidentity 90% sequence identityto to the the amino acidsequence amino acid sequenceshown shownin in

SEQ SEQ IDID NO: NO: 1. 1. In some In some embodiments, embodiments, themutant the pepD pepD mutant protein protein includesincludes a sequence a sequence having at having at

least least 95% sequenceidentity 95% sequence identity to to the the amino acid sequence amino acid sequenceshown shownin in SEQ SEQ ID NO: ID NO: 1. In1.some In some embodiments, thepepD embodiments, the pepD mutant mutant protein protein includes includes a sequence a sequence having having at least at least 99%99% sequence sequence

identity identity to tothe theamino amino acid acid sequence sequence shown inSEQ shown in SEQIDID NO:NO: 1. 1.

[0038]

[0038] InInsome some embodiments, embodiments, the the pepDpepD mutant mutant protein protein has has an an amino amino acid sequence acid sequence shown in shown in

SEQ IDNO: SEQ ID NO:1.1.

[0039]

[0039] InInsome some embodiments, embodiments, the the fadRfadR mutant mutant protein protein includes includes at least at least oneone of mutations of mutations

A140T and A140T and L171I. L171I. For example, For example, a mutation a mutation involved involved in theinfadR the fadR mutantmutant protein protein may bemay a be a A140T mutationA140T mutation A140Tor or a a mutation mutation L171I, L171I, denoted denoted as fadRor as fadRA¹ fadR fadRL171I orL171I , respectively; , respectively; or or the the

mutation involved mutation involvedinin the the fadR fadRmutant mutantprotein proteinmay maybebemutations mutations A140T A140T and L171I, and L171I, denoted denoted as as

7

A140T, L171I fadR fadR A140T, L171I . A140T

[0040]

[0040] The fadR The refers totothe fadR A140T refers thealanine alanineatat position 140140ofof position an an amino aminoacid acidsequence sequence(SEQ ID (SEQ ID

NO: 5) of the fadR protein being substituted by threonine, and the relevant activity of this protein NO: 5) of the fadR protein being substituted by threonine, and the relevant activity of this protein

mutant is tested. mutant is tested.

L171I refers to the leucine at position 171 of the amino acid sequence of the

[0041]

[0041] The fadR The fadR L171I refers to the leucine at position 171 of the amino acid sequence of the fadR proteinbeing fadR protein being substituted substituted by isoleucine, by isoleucine, and and the the relevant relevant activityactivity of this of this protein protein mutant ismutant is

tested. tested.

[0042]

[0042] InInsome some embodiments, embodiments, the the fadRfadR mutant mutant protein protein includes includes a sequence a sequence having having at least at least 80% 80%

sequence identity to sequence identity to an an amino acid sequence amino acid sequenceshown shownin in SEQ SEQ ID NO: ID NO: 2. In2.some In embodiments, some embodiments, the fadR the mutantprotein fadR mutant protein includes includes aa sequence sequencehaving havingatatleast least 85% sequence 85% sequence identitytotothe identity the amino amino acid sequence acid shownininSEQ sequence shown SEQID ID NO:NO: 2. some 2. In In some embodiments, embodiments, the fadRthe fadRprotein mutant mutant protein includes includes a a sequence havingatat least sequence having least 90% sequenceidentity 90% sequence identityto to the the amino acidsequence amino acid sequenceshown shownin in

SEQ SEQ IDID NO: NO: 2. 2. In some In some embodiments, embodiments, themutant the fadR fadR mutant proteinprotein includes includes a sequence a sequence having at having at

least least 95% sequenceidentity 95% sequence identity to to the the amino acid sequence amino acid sequenceshown shownin in SEQ SEQ ID NO: ID NO: 2. In2.some In some embodiments, thefadR embodiments, the fadRmutant mutant protein protein includes includes a sequence a sequence having having at at least99% least 99% sequence sequence identity identity

to the to the amino acid sequence amino acid shownininSEQ sequence shown SEQID ID NO:NO: 2. 2.

[0043]

[0043] InInsome some embodiments, embodiments, the the engineered engineered strain strain comprises comprises the pepD the pepD mutant mutant protein protein and the and the

fadR mutantprotein. fadR mutant protein. TheThe pepD pepD mutant mutant protein protein includes includes mutations mutations S21T,S21T, G225A,G225A, and A484, and A484,

and the and the fadR mutantprotein fadR mutant proteinincludes includesmutations mutationsA140T A140Tandand L171I. L171I.

[0044]

[0044] InInsome some embodiments, embodiments, the the pepDpepD mutant mutant protein protein has has an an amino amino acid sequence acid sequence shown in shown in

SEQ SEQ IDID NO: NO: 1, 1, andand thethe fadR fadR mutant mutant protein protein hashas an an amino amino acidacid sequence sequence shown shown in SEQinID SEQ NO: ID NO:

2. 2.

[0045] The

[0045] The engineered engineered strain strain inin thepresent the presentdisclosure disclosureis is obtained obtained by by modification modificationfrom fromthe the parent strain. parent Theparent strain. The parentstrain strain refers refers to toan anoriginal originalstrain used strain forfor used breeding. breeding. For For example, the example, the

parent strain may originate from a spontaneously mutated strain in production, or from a strain parent strain may originate from a spontaneously mutated strain in production, or from a strain

exhibiting traitsfavorable exhibiting traits favorableforfor further further research research or application, or application, such such as as growth rapid rapid growth or low or low nutritional requirements. nutritional Forexample, requirements. For example, theparent the parentstrain strainmay maybebea astrain strainthat that has has already already

undergoneother undergone othermutations mutationsorora amutator mutatorvariant variantwith withhigh highsensitivity sensitivity to to mutagens. mutagens.

[0046]

[0046] InInsome some embodiments, embodiments, the the parent parent strain strain is is selectedfrom selected from any any oneone of of Escherichia Escherichia coli, coli,

Corynebacterium glutamicum, Corynebacterium glutamicum, BacillusBacillus subtilis,subtilis, or yeast or yeast cells. cells.

[0047]

[0047] InInsome some embodiments, embodiments, the the parent parent strain strain is is selectedfrom selected from one one of of a strainIBEWQ, a strain IBEWQ,a a

mutant strain IBEWQ-62, mutant strain a mutant IBEWQ-62, a mutant strain strain IBEWQ-624, IBEWQ-624, an Escherichia an Escherichia coli Nissle1917, coli Nissle 1917,

Escherichiacoli Escherichia coli BL21, Escherichiacoli BL21, Escherichia coliHB101, HB101, Escherichia Escherichia coli coli JM109, JM109, Escherichia Escherichia coli coli

DH10B,ororEscherichia DH10B, Escherichia coli coliMG1655. MG1655.

[0048]

[0048] InInsome some embodiments, embodiments, the the modification modification includes includes enhancing enhancing an expression an expression level level of a of a pepD protein of the parent strain. pepD protein of the parent strain.

[0049]

[0049] InInsome some embodiments, embodiments, the the modification modification includes includes weakening weakening an expression an expression level level of a of a

fadR proteinofofthethe fadR protein parent parent strain. strain.

[0050]

[0050] InInsome some embodiments, embodiments, the the modification modification includes includes knocking knocking out aout a fadR fadR gene gene in a genome in a genome

of of the the engineered engineered strain strain including including the thepepD pepD mutant protein with mutant protein with mutations mutationsS21T, S21T,G225A, G225A,andand

A484 andthe A484 and thefadR fadRmutant mutant proteinwith protein with mutations mutations A140T A140T and L171I, and L171I, and replacing and replacing the promoter the promoter

of of aa gene gene encoding the pepD encoding the pepDmutant mutantprotein proteininina agenome genomeof of thetheparent parentstrain strainwith withaastrong strong promoter. promoter.

[0051] Under

[0051] Under a same a same culture culture condition, condition, a tryptophan a tryptophan production production of of thethe engineered engineered strain strain is is

increased increased compared withthe compared with theparent parentstrain. strain.

[0052] Embodiments

[0052] Embodiments of the of the present present disclosure disclosure provide provide a method a method for constructing for constructing an engineered an engineered

strain. In some strain. In someembodiments, embodiments,thethe method method comprises: comprises: obtaining obtaining the engineered the engineered strain strain by by

modifyingatat least modifying least one one of of aa pepD protein or pepD protein or aa fadR protein of fadR protein of the the parent parentstrain. strain. Under Under aa same same culture condition,a atryptophan culture condition, tryptophan production production of theof the engineered engineered strain isstrain higheristhan higher than a tryptophan a tryptophan

production of the parent strain. production of the parent strain.

[0053]

[0053] InInsome some embodiments, embodiments, the the modification modification includes includes generating generating the pepD the pepD mutant mutant protein protein by by introducing introducing a a mutation mutation at amino at an an amino acidofsite acid site theof theprotein pepD pepD ofprotein of thestrain, the parent parentwherein strain,thewherein the mutation includes at mutation includes at least least one one of ofmutations mutations S21T, G225A,andand S21T, G225A, A484K; A484K; or overexpressing or overexpressing a gene a gene

encoding the pepD encoding the pepDprotein proteinororaagene geneencoding encodingthethepepD pepD mutant mutant protein protein using using a high-copy a high-copy

plasmid as a vector; or in a genome of the parent strain, replacing the promoter of a gene plasmid as a vector; or in a genome of the parent strain, replacing the promoter of a gene

encoding the pepD encoding the pepDprotein proteinororthe thepromoter promoterofofa agene geneencoding encoding thepepD the pepD mutant mutant protein protein with with a a

strong strong promoter; or improving promoter; or improvingthe thestability stability of of mRNA transcribedfrom mRNA transcribed from a gene a gene encoding encoding the the pepD pepD

protein or protein or aa gene gene encoding the pepD encoding the mutantprotein. pepD mutant protein.

[0054]

[0054] InInsome some embodiments, embodiments, the the pepDpepD mutant mutant protein protein includes includes a sequence a sequence havinghaving at least at least 80% 80%

sequence identity to sequence identity to the the amino acid sequence amino acid shownininSEQ sequence shown SEQID ID NO:NO: 1. 1.

[0055]

[0055] InInsome some embodiments, embodiments, the the modification modification includes: includes: knocking knocking out aout a gene gene encoding encoding the the

fadR proteinininthethe fadR protein parent parent strain; strain; or or introducing introducing a mutation a mutation at anacid at an amino amino siteacid sitefadR of the of the fadR protein of protein of the the parent parentstrain, strain,wherein whereinthe mutation the mutationincludes includesatat least oneone least of of mutations A140T mutations A140T and and

L171I; or L171I; or in in aa genome ofthe genome of the parent parent strain, strain, replacing replacingthe thepromoter promoter of of aagene gene encoding the fadR encoding the fadR

protein or protein or the the promoter promoter of of aa gene gene encoding the fadR encoding the fadRmutant mutantprotein proteinwith withaaweak weakpromoter; promoter; or or

inhibiting inhibiting translation translationefficiency ororreducing efficiency stability reducing of mRNA stability of mRNA transcribed transcribed from from aa gene gene encoding encoding

the fadR the protein or fadR protein or aa gene gene encoding the fadR encoding the fadRmutant mutantprotein. protein. In In some some embodiments, embodiments, the fadR the fadR

mutantprotein mutant protein includes includes aa sequence havingatatleast sequence having least 80% sequenceidentity 80% sequence identitytotothe the amino aminoacid acid sequence shown sequence shown inin SEQ SEQ ID ID NO: NO: 2. 2.

[0056]

[0056] InInsome some embodiments, embodiments, the the modification modification includes includes following following operations. operations.

9

[0057] The

[0057] The endogenous endogenous pepDpepD and/or and/or fadR fadR proteins proteins ofstarting of the the starting strain strain areare directlymodified, directly modified, or or the the exogenous pepDand/or exogenous pepD and/orfadR fadR proteinsarearemodified proteins modified before before being being introduced introduced into into thethe

starting starting strain. strain. The modificationmethod The modification methodisisselected selectedfrom fromatatleast least one of the one of the following following

techniques. techniques.

[0058]

[0058] a.a.In Inthe the parent parent strain, strain, the themodification modification of ofthe thepepD pepD protein protein may be any may be any one oneof of the the following. following.

[0059] (a1)Introducing

[0059] (al) Introducingmutations mutations at at threeamino three amino acidsites acid sitesofof the the pepD pepDprotein, protein,including including S21T, mutations S21T, mutations S21T,G225A, G225A,andand A484K, A484K, to obtain to obtain the pepD the pepD mutant mutant protein, protein, referred referred to astopepl as pepD S21T,

G225A, G225A, A484K A484K ,, and and an an amino acid sequence amino acid sequenceofof the the pepD pepDmutant mutantprotein proteinbeing beingshown shown in in SEQSEQ ID NO: ID NO:

1. 1.

[0060] (a2)Overexpressing

[0060] (a2) Overexpressing a gene a gene encoding encoding the the pepD pepD protein protein or aor a gene gene encoding encoding the mutant the mutant

S21T,G225A, G225A,A484K A484Kusing a high-copy plasmid as the vector. protein pepD protein pepD S21T, using a high-copy plasmid as the vector.

[0061] (a3)InInaagenome

[0061] (a3) genomeof of theparent the parentstrain, strain, replacing replacing the the promoter of aa gene promoter of gene encoding encodingthe the pepDprotein pepD proteinoror the the promoter promoterofofaa gene geneencoding encodingthe themutant mutantprotein pepDS21T, proteinpepD S21T, G225A, A484K G225A, A484K with with a a strong strong promoter. promoter.

[0062] (a4)Improving

[0062] (a4) Improvingthethe stabilityofof mRNA stability mRNA transcribed transcribed from from a gene a gene encoding encoding the pepD the pepD

protein or protein or aa gene gene encoding the mutant encoding the pepDS21T, protein pepD mutant protein S21T, G225A, A484K G225A, A484K .

[0063] (a5)Any

[0063] (a5) Any manner manner or combination or combination of manners of manners capable capable of enhancing of enhancing an expression an expression level level

S21T, G225A, G225A, A484K of of aa gene gene encoding the pepD encoding the pepDprotein proteinororaa gene geneencoding encodingthe themutant mutantprotein proteinpepD pepD S21T, A484K .

[0064]

[0064] b.b.InInthe the parent parent strain, strain, the themodification modification of ofthe thefadR fadR protein proteinmay may be be any any one of the one of the

following. following.

[0065] (b1)Knocking

[0065] (b1) Knockingoutout a gene a gene encoding encoding the the fadRfadR protein protein in the in the parent parent strain. strain.

[0066] (b2)Introducing

[0066] (b2) Introducingmutations mutations at at two two amino amino acid acid sitesofofthe sites thegene geneencoding encodingthethe fadR fadR

protein, including protein, including mutations mutations A140T andL171I, A140T and L171I, to to obtainthe obtain thefadR fadRmutant mutant protein,referred protein, referredtotoasas A140T, L171I fadR fadR A140T, L171I ,, and and an an amino acid sequence amino acid sequenceofofthe the fadR fadRmutant mutantprotein proteinbeing beingshown shownin in SEQ SEQ ID ID

NO: 2. NO: 2.

[0067] (b3)InIna agenome

[0067] (b3) genomeof of thetheparent parentstrain, strain, replacing replacing the the promoter promoterofof aa gene geneencoding encodingthe the fadR proteinororthethe fadR protein promoter promoter of a of a gene gene encoding encoding theprotein the mutant mutantfadR A140T,fadRA140T, L171I protein L171I with a weak with a weak

promoter. promoter.

[0068] (b4)Inhibiting

[0068] (b4) Inhibitingthe thetranslation translation efficiency efficiency or or reducing reducing the the stability stabilityof of mRNA transcribed mRNA transcribed

from from a agene gene encoding encoding the fadR the fadR protein protein or aencoding or a gene gene encoding the mutant the mutant protein fadRprotein fadRA140T, L171I. A140T, L171I

[0069] (b5)Any

[0069] (b5) Any manner manner or combination or combination of manners of manners capable capable of weakening of weakening an expression an expression level level

of a gene of a geneencoding encodingthe the fadRfadR protein protein or aencoding or a gene gene encoding the the mutant mutant protein protein fadR L171I A140T, L171I. A140T, fadR

[0070] Further,

[0070] Further, in specific in specific embodiments embodiments of thedisclosure, of the present present disclosure, the parent the parent strain is a strain is a

tryptophan-producing strain; and the parent strain is selected from any one of Escherichia coli, tryptophan-producing strain; and the parent strain is selected from any one of Escherichia coli,

10

Corynebacterium glutamicum, Corynebacterium glutamicum, Bacillus Bacillus subtilis,yeast subtilis, yeastcells, cells, or or the the like. Whenthetheparent like. When parentstrain strain is is Escherichia coli,ititisis further Escherichia coli, furtherpreferably preferably selected selected fromfrom one one of of a strain a strain IBEWQ,IBEWQ, a mutant strain a mutant strain

IBEWQ-62, IBEWQ-62, a mutant a mutant strain strain IBEWQ-624, IBEWQ-624, an Escherichia an Escherichia coli Nissle1917, coli Nissle1917, Escherichia Escherichia coli coli BL21, Escherichiacoli BL21, Escherichia coliHB101, HB101, Escherichia Escherichia coli coli JM109, JM109, Escherichia Escherichia coli coli DH10B, DH10B, or or Escherichiacoli Escherichia coli MG1655. MG1655.

[0071]

[0071] InInsome some embodiments, embodiments, the the engineered engineered strain strain is the is the mutant mutant strain strain IBEWQ-624. IBEWQ-624. The The mutantstrain mutant strain IBEWQ-624 includes IBEWQ-624 includes a pepD a pepD mutant mutant and aand a fadR fadR mutant mutant protein, protein, the pepD the pepD mutantmutant

protein has protein has an an amino acid sequence amino acid sequenceshown shownin in SEQ SEQ ID NO: ID NO: 1, and 1, and the fadR the fadR mutant mutant protein protein has has an an amino acidsequence amino acid sequenceshown shownin in SEQ SEQ ID NO: ID NO: 2. 2.

[0072]

[0072] InInsome some embodiments, embodiments, the the obtaining obtaining the the engineered engineered strain strain includes includes culturing culturing thethe parent parent

strain strain in inculture culturemedia mediaincluding including different differentconcentrations concentrationsofoftryptophan tryptophanand andmeasuring measuring a a biomass biomass

in in each each culture culture medium; determininga agrowth medium; determining growth rateofofthe rate theparent parent strain strain based on the based on the biomass; and biomass; and

obtaining the engineered obtaining the strain capable engineered strain capable of of increasing increasing aa tryptophan tryptophan production by fermenting production by fermentingaa strain withaafaster strain with fastergrowth growth rate rate in in a culture a culture medium medium including including high-concentration high-concentration tryptophan. tryptophan.

[0073]

[0073] InInsome some embodiments, embodiments, a concentration a concentration of the of the high-concentration high-concentration tryptophan tryptophan is inisain a

range of 50 g/L to 70 g/L. range of 50 g/L to 70 g/L.

[0074]

[0074] InInspecific specificembodiments embodimentsof of thethe present present disclosure,the disclosure, theengineered engineeredstrain strainIBEWQ-624 IBEWQ-624is is

constructed by knocking constructed by knockingout outaagene geneencoding encodingthethefadR fadR proteininina agenome protein genomeof of thethe strainIBEWQ, strain IBEWQ, mutating aa gene mutating geneencoding encodingthe thepepD pepD proteininto protein intoa agene geneencoding encoding pepDS21T, pepD S21T, G225A, A484K protein, G225A, A484K protein, S21T, G225A, A484K and replacing the promoter of the gene encoding pepD and replacing the promoter of the gene encoding pepD S21T, G225A, protein with a strong A484K protein with a strong

promoterPJ23119. promoter PJ23119.

[0075] One

[0075] One of of embodiments embodiments of the of the present present disclosure disclosure provides provides a mutant a mutant protein. protein. The mutant The mutant

protein includes protein includes aa sequence havingat sequence having at least least 80% sequenceidentity 80% sequence identity to to an an amino aminoacid acidsequence sequence shown inSEQ shown in SEQID ID NO:NO: 1 or1 a orsequence a sequence having having at least at least 80%80% sequence sequence identity identity to amino to an an amino acid acid

sequence shown sequence shown inin SEQ SEQ ID ID NO: NO: 2. 2.

[0076]

[0076] InInsome some embodiments, embodiments, the the mutant mutant protein protein includes includes a pepD a pepD mutant mutant protein, protein, andpepD and the the pepD mutantprotein mutant protein includes includes at at least leastone one of ofmutations mutations S21T, S21T, G225A, andA484K. G225A, and A484K.

[0077]

[0077] InInsome some embodiments, embodiments, the the mutant mutant protein protein includes includes a fadR a fadR mutant mutant protein, protein, and and the pepD the pepD

mutantprotein mutant protein includes includes at at least leastone one of ofmutations mutations A140T andL171I. A140T and L171I.

[0078] One

[0078] One of of embodiments embodiments of the of the present present disclosure disclosure provides provides a DNA a DNA molecule. molecule. In some In some

embodiments, theDNA embodiments, the DNA molecule molecule comprises comprises a gene a gene encoding encoding the mutant the mutant protein protein of anyof anyofone of one

claims 19-21. claims 19-21.

[0079] One

[0079] One of of embodiments embodiments of the of the present present disclosure disclosure provides provides a gene a gene expression expression cassette. cassette. In In some embodiments, some embodiments, thethe gene gene expression expression cassette cassette comprises comprises the the mutant mutant protein protein described described above above

or or the the gene gene encoding the mutant encoding the mutantprotein protein described describedabove. above.

11

[0080] One

[0080] One of of embodiments embodiments of the of the present present disclosure disclosure provides provides a recombinant a recombinant vector. vector. In some In some

embodiments, therecombinant embodiments, the recombinant vector vector comprises comprises the the mutant mutant protein protein described described above above or the or the genegene

encoding the mutant encoding the mutantprotein proteindescribed describedabove. above.

[0081] One

[0081] One of of embodiments embodiments of the of the present present disclosure disclosure provides provides a use a use of an of an engineered engineered strain strain in in

increasing increasing tryptophan production. tryptophan production.

[0082]

[0082] InInsome some embodiments, embodiments, the the use use includes includes fermenting fermenting and culturing and culturing the engineered the engineered strain strain

to obtain to obtain tryptophan tryptophan with increased production. with increased production.

[0083]

[0083] InInsome some embodiments, embodiments, the the pepDpepD mutant mutant protein protein included included in theinengineered the engineered strain strain

includes includes any one of any one of the the following: following: (A1) (A1) aa protein protein having an amino having an aminoacid acidsequence sequenceshown shownin in SEQSEQ

ID NO:1;1;or ID NO: or (A2) (A2)aa protein protein derived derived from fromthe the amino aminoacid acidsequence sequence shown shown in SEQ in SEQ ID 1NO: 1 ID NO:

containing one containing one or or more more aminoamino acid substitutions, acid substitutions, deletions, deletions, and/or insertions, and/or insertions, and possessing and possessing the the same functionas same function as the the protein protein having an amino having an aminoacid acidsequence sequenceshown shown in in SEQSEQ ID NO: ID NO: 1; or1;(A3) or (A3) a a protein having protein an amino having an aminoacid acidsequence sequencewith withatatleast least 99%, 99%,95%, 95%, 90%, 90%, 85%, 85%, or 80% or 80% homology homology to to any one of any one of the the amino acid sequences amino acid sequencesdefined definedinin(A1) (A1)and and(A2), (A2),and andpossessing possessing thesame the same function function

as the proteins as the proteinsdefined definedin in (A1) (A1) and and (A2);(A2); or a(A4) or (A4) a fusion fusion protein protein obtained obtained by linkingby linking a tag a tag to the to the

N-terminusand/or N-terminus and/orC-terminus C-terminusofof any any one one of of theproteins the proteinsdefined definedinin(A1) (A1)toto(A3). (A3).

[0084]

[0084] InInsome some embodiments, embodiments, the the fadRfadR mutant mutant protein protein included included in engineered in the the engineered strain strain

includes includes any one of any one of the the following: following: (A1) a protein (A1) a protein having an amino having an aminoacid acidsequence sequenceshown shownin in SEQ SEQ

ID NO:2;2;or ID NO: or (A2) (A2)aa protein protein derived derived from fromthe the amino aminoacid acidsequence sequence shown shown in SEQ in SEQ ID 2NO: 2 ID NO:

containing one or more amino acid substitutions, deletions, and/or insertions, and possessing the containing one or more amino acid substitutions, deletions, and/or insertions, and possessing the

same functionas same function as the the protein protein having an amino having an aminoacid acidsequence sequenceshown shown in in SEQSEQ ID NO: ID NO: 2; or2;(A3) or (A3) a a protein having protein an amino having an aminoacid acidsequence sequencewith withatatleast least 99%, 99%,95%, 95%, 90%, 90%, 85%, 85%, or 80% or 80% homology homology to to any one of any one of the the amino acid sequences amino acid sequencesdefined definedinin(A1) (A1)and and(A2), (A2),and andpossessing possessing thesame the same function function

as the proteins as the proteinsdefined definedin in (A1) (A1) and and (A2);(A2); or a(A4) or (A4) a fusion fusion protein protein obtained obtained by linkingby linking a tag a tag to the to the

N-terminusand/or N-terminus and/orC-terminus C-terminusofof any any one one of of theproteins the proteinsdefined definedinin(A1) (A1)toto(A3). (A3).

[0085]

[0085] InInsome some embodiments, embodiments, the the genegene encoding encoding the pepD the pepD mutantmutant protein protein includes includes a DNA a DNA

molecule havingatatleast molecule having least 99%, 95%,90%, 99%, 95%, 90%, 85%, 85%, or 80% or 80% homology homology to asequence to a DNA DNA sequence defined defined

by aa gene by encodingthe gene encoding pepDS21T, thepepD S21T, G225A, A484K G225A, A484K mutant protein. mutant protein.

[0086]

[0086] InInsome some embodiments, embodiments, the the genegene encoding encoding the fadR the fadR mutant mutant protein protein includes includes a DNA a DNA

molecule havingatatleast molecule having least 99%, 95%,90%, 99%, 95%, 90%, 85%, 85%, or 80% or 80% homology homology to asequence to a DNA DNA sequence defined defined

by aa gene by encodingthe gene encoding fadRA140T, thefadR L171I A140T, L171I mutantprotein. mutant protein.

[0087] The

[0087] The present present disclosureprovides disclosure provides any any oneone of of thethe following following biomaterials,which biomaterials, which maymay be be

used to used to increase increase tryptophan production. tryptophan production.

[0088] (I)Protein:

[0088] (I) pepDS21T, Protein: pepD S21T, G225A, A484K G225A, A484K mutantprotein mutant protein and/or fadRA140T, and/or fadR L171I A140T, L171I mutantprotein. mutant protein.

[0089] (II)Gene:

[0089] (II) Gene:a agene geneencoding encoding pepDS21T, pepD S21T, G225A, A484K G225A, A484K mutant protein and/or mutant protein and/or aa gene encoding gene encoding

12

A140T, L171I fadR fadR A140T, L171I mutantprotein. mutant protein. S21T,

[0090] (III) Expression

[0090] (III) Expressioncassette: cassette: an an expression expression cassette cassette including a gene including a encodingpepD gene encoding pepDS21T,

G225A, G225A, A484K A140T, L171I L171I A484K mutantprotein mutant protein and/or and/or aa gene gene encoding encodingfadR fadRA140T, mutantprotein, mutant protein, or or an an

expression cassette including expression cassette including a a DNA fragment DNA fragment of of thegene the gene encoding encoding thethe pepDS21T, pepD S21T, G225A, A484K G225A, A484K

A140T, L171I mutantprotein mutant protein and/or and/or aa DNA DNA fragment fragment of of thethe gene gene encoding encoding the the fadR fadR A140T, L171I mutantprotein. mutant protein.

[0091] (IV)Recombinant

[0091] (IV) Recombinant vector: vector: a recombinant a recombinant vector vector including including a gene a gene encoding encoding S21T, S21T, pep. pepD

G225A, G225A, A484K A484K mutantprotein mutant protein and/or and/or aa gene gene encoding fadRA140T, encodingfadR L171I mutant protein, or a A140T, L171I mutant protein, or a S21T, G225A, A484K recombinantvector recombinant vectorincluding includinga aDNA DNA fragment fragment of the of the gene gene encoding encoding the pepDS21T, the pepD G225A, A484K

A140T, L171I L171I mutantprotein mutant protein and/or and/or aa DNA DNA fragment fragment of of thethe gene gene encoding encoding the the fadRA140T, fadR mutantprotein. mutant protein. S21T, G225A,

[0092] (V)Recombinant

[0092] (V) Recombinant strain: strain: a recombinant a recombinant strain strain including including a gene a gene encoding encoding pepDpepDS21T, G225A,

A484K mutant protein and/or a gene encoding fadR A140T, L171I A140T,mutant L171I protein, or a recombinant A484K mutant protein and/or a gene encoding fadR mutant protein, or a recombinant strain strain including including aaDNA fragmentofofthe DNA fragment thegene geneencoding encodingthethe pepDS21T, pepD S21T, G225A, A484K G225A, A484K mutantprotein mutant protein A140T, L171I and/or aa DNA and/or fragment DNA fragment of of thethegene gene encoding encoding thethe fadR fadR A140T, L171I mutantprotein. mutant protein.

[0093]

[0093] (a)(a) A use A use of a of a biomaterial biomaterial in increasing in increasing tryptophan tryptophan productionproduction of a parent of a parent strain. strain.

[0094] (b)A Ause

[0094] (b) useofofa abiomaterial biomaterialininproducing producingtryptophan. tryptophan.

[0095]

[0095] (c)(c) A use A use of pepD S21T, S21T, of pepD G225A, G225A, A484KA484K mutant mutant protein protein or fadRor A140T,mutant fadRL171I A140T, L171I protein in mutant protein in increasing tryptophan increasing tryptophan production production of a parent of a parent strain.strain.

S21T, G225A, A484K A140T, L171I L171I

[0096]

[0096] (d) (d) A use of pepDS21T, use of pepD G225A, A484K mutantprotein mutant protein or or fadR fadRA140T, mutantprotein mutant protein in in A producingtryptophan. producing tryptophan.

Embodiments Embodiments

[0097] The

[0097] The following following provides provides a detailed a detailed descriptionofofspecific description specificembodiments embodimentsof of thethe present present

disclosure. However, disclosure. However,it it should should bebe understood understood that that thethescope scope ofof protectionofofthe protection thepresent present disclosure disclosure isisnot notlimited limitedto tothese these specific specific embodiments. embodiments.

[0098]

[0098] InInthe thepresent presentdisclosure, disclosure, unless unless otherwise otherwise specified, specified, the the experimental methodsused experimental methods usedare are conventional methods,and conventional methods, andthe thematerials materialsand andreagents reagentsare arecommercially commercially available. available.

Embodiment 1 Screening Embodiment 1 Screening for afor a tolerant tolerant strain strain with with accelerated accelerated growth growth rate inrate in tryptophan tryptophan

fermentation broth fermentation broth

[0099] Fusion

[0099] Fusion of of cytidinedeaminase cytidine deaminase with with thethesubunit α subunit of Escherichia of Escherichia colicoli RNARNA polymerase polymerase

can acceleratemutations can accelerate mutations to levels to levels that that support support efficient efficient adaptive adaptive evolution evolution in Escherichia in Escherichia coli coli without reducing cell viability. without reducing cell viability.

[0100] Primers RNAP

[0100] Primers RNAP-F/R α-F/R andand CDA-F/R CDA-F/R werewere usedused to amplify to amplify a agene genesequence sequenceencoding encoding the the αsubunit subunitofofEscherichia Escherichiacoli coli RNA RNA polymerase polymerase and and a gene a gene sequence sequence encoding encoding the cytidine the cytidine

deaminase ofaagenome deaminase of genomeof of theparent the parentstrain, strain, respectively. respectively. Then, Then,twotwo endend sequences sequences werewere fused fused

and expressedusing and expressed usingthe the primers primersRNAP RNAP α-F CDA-R, -F and and CDA-R, digested digested with restriction with restriction enzymes enzymes

13

EcoRI andNcol, EcoRI and NcoI,and andthen thenligated ligatedinto intothe the temperature-sensitive temperature-sensitive plasmid plasmidpKD46 pKD46 (GenBank (GenBank

accession no.: accession no.: MF287367) thathas MF287367) that hasbeen been digestedwith digested with thesame the same enzymes, enzymes, to obtain to obtain thethe plasmid plasmid

denoted as denoted as pKAP, pKAP,and and theplasmid the plasmid pKAP pKAP included included an arabinose-inducible an arabinose-inducible promoter promoter regulating regulating

the expression the expression of of the the gene gene of of the the α subunit subunit of of Escherichia coli RNA Escherichia coli polymerase RNA polymerase andand thethe gene gene of of the cytidine the cytidine deaminase. deaminase.

[0101] The

[0101] The primer primer sequences sequences usedused for for constructing constructing thethe plasmid plasmid pKAPpKAP was was as as follows: follows:

[0102] RNAP

[0102] RNAP -F:α-F: GAATTCatgcagggttctgtgacag; GAATTCatgcagggttctgtgacag; SEQ SEQ ID 7. ID NO: NO: 7.

[0103]

[0103] RNAP RNAP α-R: -R: CGATCCGCCACCGCCAGAGCCACCTCCGCCctcgtcagcgatgcttgccggtg; CGATCCGCCACCGCCAGAGCCACCTCCGCCctcgtcagcgatgctgccggtg: SEQ IDNO: SEQ ID NO:8.8.

[0104]

[0104] CDA-F: CDA-F:GGCGGAGGTGGCTCTGGCGGTGGCGGATCGcatccacgttttcaaaccgc; GGCGGAGGTGGCTCTGGCGGTGGCGGATCGcatccacgtttcaaccgc; SEQSEQ ID NO:9.9. ID NO:

[0105] CDA-R:

[0105] CDA-R: CCATGGttaagcgagaagcactcgg; CCATGGttaagcgagaagcactcgg; SEQ SEQ ID 10. ID NO: NO: 10.

Embodiment Embodiment 22

[0106] (1)Construction

[0106] (1) Constructionofofa aparent parentstrain strain IBEWQ IBEWQ

[0107] The

[0107] The parent parent strainIBEWQ strain IBEWQ was derived was derived from from Escherichia Escherichia coli W3110 coli W3110 (competent (competent cells ofcells of

whichwere which werecommercially commercially available available from from various various biological biological reagent reagent suppliers).Specifically, suppliers). Specifically, at at the tnaA the locus, the tnaA locus, trpES40F, the trpE S40F,M1293T DCBA M1293T DCBA gene gene was was expressed in tandem expressed in underthe tandem under thecontrol controlof of the the tac promoter, at the trpR locus, AroF^P¹L, ^P148L, tac promoter, at the trpR locus, AroF Q1521, Q152I, N8K ,N8K , Aro GL76V,D146N AroLV, P150L, P150L,, D146N , tktA, and ppsA genes tktA, and ppsA genes

derived from derived fromEscherichia Escherichiacoli coli K-12 K-12were wereexpressed, expressed, and and at at thetyrR the tyrRlocus, locus,the the SerA SerAgene genederived derived from Bacillus subtilis from Bacillus subtilis was was expressed. expressed. AtAt thesame the same time, time, thepromoters the promoters of of thetyrA the tyrA and and pheA pheA

genes were genes werereplaced replacedwith withthe thepromoter promoterPJ23114. PJ23114.

[0108] (2)Acquisition

[0108] (2) Acquisitionofofa amutant mutantstrain strainIBEWQ-62 IBEWQ-62

[0109] The

[0109] The plasmid plasmid pKAP pKAP was electrotransformed was electrotransformed intostrain into the the strain IBEWQ IBEWQ to generate to generate a strain a strain

IBEWQ-pKAP. IBEWQ-pKAP. The strain The strain IBEWQ-pKAP IBEWQ-pKAP wasasused was used theasparent the parent strain strain and and continuously continuously

subculture subculture and evolution were and evolution wereperformed performedininseed seedmedia media (supplemented (supplemented withwith ampicillin ampicillin

resistance) including resistance) including different differentconcentrations concentrationsof oftryptophan. After 12 tryptophan. After 12 hours hoursof of incubation, incubation, each each seed mediumwaswas seed medium diluted diluted twofold, twofold, and and thethe biomass biomass in in each each seed seed medium medium was measured was measured

individually. individually.

Table11Biomass Table Biomass measurement measurement valuesvalues under under different different concentrations concentrations of tryptophan of tryptophan

Concentration of tryptophan Concentration of tryptophaninin Biomass OD Biomass (0 OD600(0 Biomass OD Biomass (15 OD600(15 culture culture medium (g/L) medium (g/L) mML-arabinose) mM L-arabinose) mML-arabinose) mM L-arabinose) 30 30 0.69 0.69 0.67 0.67

40 40 0.56 0.56 0.55 0.55

14

50 0.48 0.48 0.48 0.48

60 60 0.27 0.27 0.36 0.36

70 70 0.10 0.10 0.30 0.30

[0110]

[0110] A A strainexhibiting strain exhibitingaccelerated acceleratedgrowth growthrate rateinin aa culture culture medium includinghigh- medium including high- concentration tryptophanwas concentration tryptophan wascultured culturedininaa fermentation fermentationmedium mediumat at 3737 °C °C to to eliminatethe eliminate the plasmidpKAP. plasmid pKAP. After After plating plating and and colony colony purification, purification, a mutant a mutant strain strain with with significantly significantly

improved tryptophanproduction improved tryptophan production was was designated designated as as IBEWQ-62. IBEWQ-62.

[0111] Composition

[0111] Composition of aofseed a seed medium: medium: 2.4 g/L 2.4 g/L K2HPO K2HPO4, 9.6 4, g/L9.6KHPO, g/L KH PO4,yeast 15 2g/L 15 g/L yeast extract, extract,

10 g/L g/L rice rice bran, bran,5.0 5.0g/L g/L(NH 4)2SO (NH)SO, 4, 1.0g/L 1.0g/L MgSOand MgSO·7HO, 4·7H20 2O, andglucose, g/L 20 g/L natural glucose,pH. natural pH.

[0112] Composition

[0112] Composition of aofshake a shake flask flask fermentation fermentation medium: medium: 20 glucose, 20 g/L g/L glucose, 3.0 yeast 3.0 g/L g/L yeast extract extract powder, 30 g/L powder, 30 g/L rice rice bran, bran, 1.6 1.6 g/L g/L (NH 4)2SO (NH)SO, 4, 2.0 2.0 g/Lg/L citricacid, citric acid,5.6 5.6g/L g/LKHPO, K2HPO2.04,g/L 2.0 g/L MgSO 4·7H2O, MgSO·7HO, 80 80 mg/L mg/L FeSO4·7H24.0 FeSO·7HO, O, 4.0 mg/L mg/L CoCl2·6H20.6 CoCl·6HO, O, 0.6 mg/L mg/L CuSO CuSO 4·5H6.5 5HO, 2O, 6.5 mg/Lmg/L ZnSO4·7H220 ZnSO·7HO, O, mg/L 20 mg/L Na24.5 NaSO, SO4mg/L , 4.5 mg/L MnSO MnSO·HO, 4·H and 2O, 20 andcalcium g/L 20 g/L calcium carbonate, carbonate, pH=7.2.pH=7.2.

Embodiment Embodiment 3 3 Tryptophan Tryptophan produced produced by by fermentation fermentation of of mutant mutant strainsand strains and its genomic its genomic sequencing analysis sequencing analysis

[0113] The

[0113] The parent parent strainIBEWQ strain IBEWQ and mutant and the the mutant strain strain IBEWQ-62 IBEWQ-62 were subjected were subjected to to fermentation. First,each fermentation. First, eachstrain strain preserved preserved in in glycerol glycerol was revived on was revived on slant slant agar, agar, and and then then

transferred into transferred intothe theseed seedmedium to culture medium to culture IBEWQ IBEWQ andand IBEWQ-62 IBEWQ-62 overnight. overnight. The obtained The obtained

seeds of IBEWQ seeds of IBEWQ andand IBEWQ-62 IBEWQ-62 were inoculated were inoculated into into 500 mL500 mLflasks shake shakecontaining flasks containing 100 mL 100 mL

of fermentation of medium,followed fermentation medium, followed by by cultivationatat220 cultivation 220rpm rpm and and 37°C. 37°C. Samples Samples were collected were collected

to measure to residual glucose measure residual glucose content content and andtryptophan tryptophanproduction. production.After After 45 45 hours hours of fermentation, of fermentation,

the tryptophan the productionlevel tryptophan production level of of IBEWQ-62 IBEWQ-62 waswas significantly significantly higher higher than than that that ofof theparent the parent strain. strain.

[0114] The

[0114] The genome genome of the of the mutant mutant strain strain IBEWQ-62 IBEWQ-62 was extracted was extracted and subjected and subjected to whole- to whole-

genome sequencing. genome sequencing. A comparison A comparison of genome of genome analysis analysis revealed revealed that mutations that point point mutations occurred occurred

in in the the IBEWQ-62 genome IBEWQ-62 genome compared compared to thetoparent the parent strain, strain, and and the the mutations mutations leading leading to amino to amino acid acid

mutations were mutations weresummarized summarizedin in Table Table 2. 2. Table 22 Mutations Table Mutations in in the thegenome genome of of IBEWQ-62 compared IBEWQ-62 compared to to thewild-type the wild-typestrain strain

Gene Gene Mutation Mutation Sequence after mutation Sequence after mutation Sequence beforemutation Sequence before mutation name name Mutated Mutated S21T, S21T, MSELSQLSPQPLWDIFAKICT MSELSQLSPQPLWDIFAKICT MSELSQLSPQPLWDIFAKICsIPH MSELSQLSPQPLWDIFAKICsIPH pepD pepD G225A, G225A, IPHPSYHEEQLAEYIVGWAK IPHPSYHEEQLAEYIVGWAK PSYHEEQLAEYIVGWAKEKGFH PSYHEEQLAEYIVGWAKEKGFH gene gene and and EKGFHVERDQVGNILIRKPA EKGFHVERDQVGNILIRKPA VERDQVGNILIRKPATAGMENR VERDQVGNILIRKPATAGMENR 15

A484K A484K TAGMENRKPVVLQAHLDM KPVVLQAHLDMVPQKNNDTVH KPVVLQAHLDMVPQKNNDTVH TAGMENRKPVVLQAHLDM VPQKNNDTVHDFTKDPIQPY DFTKDPIQPYIDGEWVKARGTTL VPQKNNDTVHDFTKDPIQPY DFTKDPIQPYIDGEWVKARGTTL IDGEWVKARGTTLGADNGI IDGEWVKARGTTLGADNGI GADNGIGMASALAVLADENVV GADNGIGMASALAVLADENVV GMASALAVLADENVVHGPL HGPLEVLLTMTEEAGMDGAFGL GMASALAVLADENVVHGPL HGPLEVLLTMTEEAGMDGAFGL EVLLTMTEEAGMDGAFGLQ EVLLTMTEEAGMDGAFGLQ QGNWLQADILINTDSEEEGEIYM QGNWLQADILINTDSEEEGEIYM GNWLQADILINTDSEEEGEIY GCAGGIDFTSNLHLDREAVPAGF GNWLQADILINTDSEEEGEIY GCAGGIDFTSNLHLDREAVPAGF MGCAGGIDFTSNLHLDREA MGCAGGIDFTSNLHLDREA ETFKLTLKGLKGGHSGGEIHVGL ETFKLTLKGLKGGHSGGEIHVGL VPAGFETFKLTLKGLKGGHS gNANKLLVRFLAGHAEELDLRLI VPAGFETFKLTLKGLKGGHS gNANKLLVRFLAGHAEELDLRLI GGEIHVGLaNANKLLVRFLA GGEIHVGLaNANKLLVRFLA DFNGGTLRNAIPREAFATIAVAA DFNGGTLRNAIPREAFATIAVAA GHAEELDLRLIDFNGGTLRN GHAEELDLRLIDFNGGTLRN DKVDVLKSLVNTYQEILKNELA DKVDVLKSLVNTYQEILKNELA AIPREAFATIAVAADKVDVL AIPREAFATIAVAADKVDVL EKEKNLALLLDSVANDKAALIA EKEKNLALLLDSVANDKAALIA KSLVNTYQEILKNELAEKEK KSLVNTYQEILKNELAEKEK KSRDTFIRLLNATPNGVIRNSDV KSRDTFIRLLNATPNGVIRNSDV NLALLLDSVANDKAALIAKS AKGVVETSLNVGVVTMTDNNV NLALLLDSVANDKAALIAKS AKGVVETSLNVGVVTMTDNNV RDTFIRLLNATPNGVIRNSD RDTFIRLLNATPNGVIRNSD EIHCLIRSLIDSGKDYVVSMLDSL EIHCLIRSLIDSGKDYVVSMLDSL VAKGVVETSLNVGVVTMTD GKLAGAKTEAKGAYPGWQPDA VAKGVVETSLNVGVVTMTD GKLAGAKTEAKGAYPGWQPDA NNVEIHCLIRSLIDSGKDYVV NSPVMHLVRETYQRLFNKTPNI NNVEIHCLIRSLIDSGKDYVV NSPVMHLVRETYQRLFNKTPNI SMLDSLGKLAGAKTEAKGA SMLDSLGKLAGAKTEAKGA QIIHAGLECGLFKKPYPEMDMVS QIHAGLECGLFKKPYPEMDMVS YPGWQPDANSPVMHLVRET IGPTITGPHSPDEQVHIESVGHY YPGWQPDANSPVMHLVRET IGPTITGPHSPDEQVHIESVGHY YQRLFNKTPNIQIIHAGLECG YQRLFNKTPNIQIIHAGLECG WTLLTELLKEIPaK(SEQ WTLLTELLKEIPaK (SEQID ID NO: NO: LFKKPYPEMDMVSIGPTITG LFKKPYPEMDMVSIGPTITG 4) 4)

PHSPDEQVHIESVGHYWTLL PHSPDEQVHIESVGHYWTLL TELLKEIPKK TELLKEIPKK (SEQ (SEQ IDIDNO: NO:1)1) Mutated Mutated A140T A140T MVIKAQSPAGFAEEYIIESIW MVIKAQSPAGFAEEYIIESIW MVIKAQSPAGFAEEYIIESIW MVIKAQSPAGFAEEYIIESIW fadR gene and fadR gene and NNRFPPGTILPAERELSELIGa NNRFPPGTILPAERELSELIGa NNRFPPGTILPAERELSELIGaTRT NNRFPPGTILPAERELSELIGaTRT L171I L171I TRTTLREVLQRLARDGWLTI TRTTLREVLQRLARDGWLTI TLREVLQRLARDGWLTIQHGKP TLREVLQRLARDGWLTIQHGKP QHGKPTKVNNFWETSGLNIL TKVNNFWETSGLNILETLARLD QHGKPTKVNNFWETSGLNIL TKVNNFWETSGLNILETLARLD ETLARLDHESVPQLIDNLLS ETLARLDHESVPQLIDNLLS HESVPQLIDNLLSVRTNISTIFIRT HESVPQLIDNLLSVRTNISTIFIRT VRTNISTIFIRTAFRQHPDKA VRTNISTIFIRTAFRQHPDKA AFRQHPDKAQEVLATANEVAD AFRQHPDKAQEVLATANEVAD QEVLATANEVADHADtFAEL HADaFAELDYNIFRGLAFASGNP QEVLATANEVADHADtFAEL HADaFAELDYNIFRGLAFASGNP DYNIFRGLAFASGNPIYGLIL DYNIFRGLAFASGNPIYGLIL IYGLILNGMKGlYTRIGRHYFAN IYGLILNGMKGIYTRIGRHYFAN NGMKGIYTRIGRHYFANPEA PEARSLALGFYHKLSALCSEGAH NGMKGIYTRIGRHYFANPEA PEARSLALGFYHKLSALCSEGAH

16

RSLALGFYHKLSALCSEGAH DQVYETVRRYGHESGEIWHRM RSLALGFYHKLSALCSEGAH DQVYETVRRYGHESGEIWH DQVYETVRRYGHESGEIWH QKNLPGDLAIQGR QKNLPGDLAIQGR (SEQ (SEQ IDIDNO: NO: RMQKNLPGDLAIQGR RMQKNLPGDLAIQGR (SEQ (SEQ 5) 5)

ID ID NO: 2) NO: 2)

Mutated Mutated V94L and MAISIKTPEDIEKMRVAGRL V94L and MAISIKTPEDIEKMRVAGRL MAISIKTPEDIEKMRVAGRLAAE MAISIKTPEDIEKMRVAGRLAAE map gene map gene Q182N Q182N AAEVLEMIEPYVKPGVSTGE AAEVLEMIEPYVKPGVSTGE VLEMIEPYVKPGVSTGELDRICN VLEMIEPYVKPGVSTGELDRICN LDRICNDYIVNEQHAVSACL LDRICNDYIVNEQHAVSACL DYIVNEQHAVSACLGYHGYPKS DYIVNEQHAVSACLGYHGYPKS GYHGYPKSVCISINEVVCHG GYHGYPKSVCISINEVVCHG VCISINEVVCHGIPDDAKLLKDG VCISINEVVCHGIPDDAKLLKDG IPDDAKLLKDGDIlNIDVTVI IPDDAKLLKDGDINIDVTVI DIVNIDVTVIKDGFHGDTSKMFI DIVNIDVTVIKDGFHGDTSKMFI KDGFHGDTSKMFIVGKPTIM VGKPTIMGERLCRITQESLYLAL KDGFHGDTSKMFIVGKPTIM VGKPTIMGERLCRITQESLYLAL GERLCRITQESLYLALRMVK GERLCRITQESLYLALRMVK RMVKPGINLREIGAAIQKFVEAE RMVKPGINLREIGAAIQKFVEAE PGINLREIGAAIQKFVEAEGF PGINLREIGAAIQKFVEAEGF GFSVVREYCGHGIGRGFHEEPQ GFSVVREYCGHGIGRGFHEEPQ SVVREYCGHGIGRGFHEEPn SVVREYCGHGIGRGFHEEPn VLHYDSRETNVVLKPGMTFTIEP VLHYDSRETNVVLKPGMTFTIEP VLHYDSRETNVVLKPGMTF VLHYDSRETNVVLKPGMTF MVNAGKKEIRTMKDGWTVKTK MVNAGKKEIRTMKDGWTVKTK TIEPMVNAGKKEIRTMKDG TIEPMVNAGKKEIRTMKDG DRSLSAQYEHTIVVTDNGCEILT DRSLSAQYEHTIVVTDNGCEILT WTVKTKDRSLSAQYEHTIV WTVKTKDRSLSAQYEHTIV LRKDDTIPAIISHDE LRKDDTIPAIISHDE (SEQ (SEQ ID ID NO: NO:

VTDNGCEILTLRKDDTIPAIIS VIDNGCEILTLRKDDTIPAIIS 6) 6)

HDE (SEQIDIDNO: HDE (SEQ NO: 3)3)

[0115] The

[0115] The mutation mutation sitesofofthe sites thegenes genespepD, pepD, fadR, fadR, andand mapmap are are as follows. as follows.

[0116] The

[0116] The mutated mutated pepD pepD gene, gene, denoted denoted as pepDS21T,G225A,A484Kincludes as pepDS21T,G225A,,A484K, , includes threemutations three mutations S21T, S21T,

G225A, and A484K. G225A, and A484K.

[0117] The

[0117] The mutated mutated fadR fadR gene, gene, denoted denoted as fadRA140T,L171I as fadRA¹,L¹¹, , includes includes two mutations two mutations A140T and A140T and

L171I. L171I.

V94L,Q182N

[0118]

[0118] The The mutated mutatedmap mapgene, denoted gene, as map denoted as includes , includes two mutations two mutations V94LV94L and and Q182N. Q182N.

Embodiment Embodiment 4 4 Impact Impact ofofmutated mutated genesononfermentation genes fermentationperformance performance

[0119]

[0119] InInananoriginal originalstrain, strain, the the promoters promoters of of the the amino acid sequence amino acid sequencemutated mutatedgenes genes(fadR, (fadR, pepD,and pepD, andmap map genes) genes) were were individually individually replaced replaced with with promoters promoters of different of different strengths,and strengths, andthe the impact of each impact of each mutant mutantononvaline valineproduction productionininfermentation fermentationwas wascompared. compared.

[0120] Following

[0120] Following standard standard gene gene editing editing procedures, procedures, thethe promoters promoters of the of the genes genes pepD, pepD, fadR, fadR, and and

mapinin the map the strain strain IBEWQ were IBEWQ were individually individually replaced replaced with with a strong a strong promoter promoter PJ23119. PJ23119.

Similarly, Similarly, following following standard standard gene editing procedures, gene editing the promoters procedures, the of the promoters of the genes pepD,fadR, genes pepD, fadR,

17 and mapwere and map werereplaced replacedwith witha aweak weak promoter promoter PJ23114. PJ23114. The correctly The correctly verified verified strains strains afterafter construction were construction activated in were activated in the the seed seed medium for1212toto 16 medium for 16hours hoursand andwere weretransferred transferredinto into the the fermentation medium fermentation medium thenext the nextday. day.

[0121] Afterthe

[0121] After thefermentation fermentationwas was completed, completed, thethe tryptophan tryptophan production production of each of each production production

strain strain was was shown in Table shown in Table3. 3. Table 33 Table

Modifiedprotein Modified protein IBEWQ IBEWQ IBEWQ-62 IBEWQ-62 PJ23119 PJ23119 PJ23114 PJ23114

fadR fadR 3.43±0.68g/L 3.43±0.68 g/L 4.18±0.15g/L 4.18±0.15 g/L pepD pepD 3.87±0.11 g/L 4.53±0.30 3.87±0.11 g/L 4.53±0.30g/L g/L 4.06±0.69 4.06±0.69g/L g/L 3.68±0.80 g/L 3.68±0.80 g/L

map map 3.93±0.33 g/L 3.71±0.06 3.93±0.33 g/L g/L 3.71±0.06 g/L

[0122] Based

[0122] Based on on thethe fermentation fermentation results,weakening results, weakeningof of thethe fadR fadR mutant mutant genegene significantly significantly

contributes to contributes to an an increase increase in intryptophan tryptophan production, production, whereas whereas strengthening the pepD strengthening the mutantgene pepD mutant gene is is beneficial beneficialtotoincrease tryptophan increase tryptophanyield, yield,and thethe and map mapmutant mutant has has no no noticeable noticeable impact impact on on

tryptophan production. tryptophan production.

Embodiment Embodiment 5:5:Overexpression Overexpressionofofmutated mutatedgenes genesororconstruction construction of of weakened plasmids weakened plasmids

A140T,L171I

[0123]

[0123] InInthe themutant mutantstrain strainIBEWQ-62, IBEWQ-62,the the promoter promoter of the of the fadRfadR A140T,L1711 gene wasreplaced gene was replaced by PJ23114, by PJ23114,denoted denotedasasa astrain strain IBEWQ-621. IBEWQ-621.

[0124] The

[0124] The promoter promoter of of thethe pepDS21T, pepD S21T, G225A, A484K G225A, A484K gene wasreplaced gene was replacedbybyPJ23119, PJ23119,denoted denoted as as a a

strain strainIBEWQ-622. IBEWQ-622. A140T,L171I

[0125] The

[0125] The fadR fadR A140T,L1711 gene wasknocked gene was knocked outinina agenome out genomeof of thethe strainIBEWQ-622, strain IBEWQ-622, denoted as denoted as aa strain strain IBEWQ-623. IBEWQ-623.

[0126] The

[0126] The tryptophan tryptophan production production results results areare shown shown in Table in Table 1. 1. It isIt evident is evident that that thetheenhanced enhanced expression of the expression of the pepD genesignificantly pepD gene significantly improves improvesthe thefermentation fermentationperformance, performance, while while

weakeningororeven weakening evencompletely completely knocking knocking out out thethe fadR fadR gene gene yields yields the the best best improvement improvement in in fermentation performance.Based fermentation performance. Based on this, on this, a combined a combined strain strain IBEWQ-624 IBEWQ-624 was constructed was constructed by by knockingout knocking outthe the fadR fadRgene geneininthe the genome genomeof of thestrain the strainIBEWQ-62 IBEWQ-62and and replacing replacing the the promoter promoter

S21T,G225A,A484K of of the the pepD pepD S21T,G225A,A484K gene by PJ23119. gene by PJ23119.

[0127] The

[0127] The fermentation fermentation level level ofof thestrain the strainIBEWQ-624 IBEWQ-624 reaches reaches 4.91 4.91 ± 0.28 ± 0.28 g/L, g/L, withwith a a glucose-to-tryptophanyield glucose-to-tryptophan yield of of 24.5%. 24.5%.These These results results demonstrate demonstrate thatthat weakening weakening the fadR the fadR gene gene

is is beneficial beneficialfor forimproving improving the thefermentation fermentation performance, with aa complete performance, with completeknockout knockoutofofthe thefadR fadR gene representing gene representing thethe most most effective effective strategy. strategy.

Embodiment Embodiment 6 6 Scale-upvalidation Scale-up validation

[0128] The

[0128] The parent parent strainIBEWQ strain IBEWQ and mutant and the the mutant strain strain IBEWQ-624 IBEWQ-624 were inoculated were inoculated into 500into 500

18 mLshake mL shakeflasks flaskscontaining containing100 100mLmL of of seed seed medium, medium, respectively, respectively, and and cultured cultured at 37 °C at 37 °C andand 200 200 rpmfor rpm for 12 12 to to 16 hours, with 16 hours, with an an OD 600 values OD values of 11-13. of 11-13. The cultured The cultured seed solution seed solution was was inoculated intoa a5 5L L inoculated into fermenter fermenter at a at a 10% 10% (v/v) (v/v) inoculation inoculation volume, volume, with an with an initial initialrate aeration aeration of rate of 1.5 1.5 vvm andananinitial vvm and initial agitation agitationspeed speedof of400 400rpm. During rpm. During fermentation, fermentation, 25% 25% ammonia ammonia waterwater was fed was fed into into the the fermenter fermenter to to maintain maintain the the pH at 7.0. pH at Thefermentation 7.0. The fermentationtemperature temperature waswas controlled at 37 ± 0.5°C, and the agitation speed and aeration rate were manually adjusted to controlled at 37 ± 0.5°C, and the agitation speed and aeration rate were manually adjusted to maintain the maintain the dissolved dissolved oxygen oxygenlevel levelin in aa range of 20% range of to30%. 20% to 30%.After After inoculation inoculation forfor approximately approximately 66hours, hours,aa sharp sharp increase increase in in dissolved dissolved oxygen level was oxygen level wasobserved, observed,indicating indicating depletion depletion of of the the initial initialglucose. glucose. The automatic feeding The automatic feedingmode modewaswas then then activated,and activated, and800800 g/L g/L of of glucose wasfed glucose was fedto to maintain maintain the the glucose glucose concentration concentrationin in the the fermentation broth below fermentation broth below11g/L. g/L.

[0129] After1616hours

[0129] After hoursofoffermentation, fermentation,samples samples were were taken taken every every 2 to 2 to 4 hours 4 hours forfor analysis.At At analysis.

48 hours, 48 hours, the the tryptophan production of tryptophan production of the the parent parent strain strainIBEWQ reached IBEWQ reached 42.12 ± 4.61 g/L, 42.12 ± 4.61 g/L, a a glucose-to-tryptophanyield glucose-to-tryptophan yield reached reached19.1%, 19.1%,and andthe thetryptophan tryptophanproduction production of of themutant the mutant strain strain

IBEWQ-624 IBEWQ-624 reached reached 62.38 ± 5.80 g/L 62.38 ± 5.80 g/L and and the glucose-to-tryptophan the glucose-to-tryptophan yieldyield reached reached 24.1%. 24.1%.

Embodiment 7 Use Embodiment 7 Use of modification of modification manners manners in strains in other other strains

[0130] Taking

[0130] Taking Escherichia Escherichia coli coli Nissle1917 Nissle 1917 as as a a parentstrain, parent strain, in in the the genome ofthe genome of the parent parent strain, strain,a afadR fadRgene gene was was knocked out, aa pepD knocked out, pepDgene genewas was mutated mutated to to pepDS21T,G225A,A484K,, and pepDS21T,G225A,A484K, the and the

promoterofof the promoter the pepD pepDgene genewas was replaced replaced by by PJ23119 PJ23119 to obtain to obtain a recombinant a recombinant strain strain N-RD. N-RD.

After 40 After hours of 40 hours of shake flask fermentation, shake flask fermentation, 0.97 ± 0.06 g/L 0.97 ± 0.06 g/L of of tryptophan tryptophan was measuredininthe was measured the fermentation broth fermentation broth of the of the parent parent strain strain Escherichia Escherichia coli Nissle coli Nissle 1917, 1917, while while 1.28 1.28 ± 0.03 g/L ± 0.03 g/L of of tryptophan was tryptophan wasmeasured measuredin in thefermentation the fermentationbroth brothofofthe therecombinant recombinant strain,representing strain, representingaa 1.32-fold increaseininthethe 1.32-fold increase tryptophan tryptophan production. production.

[0131] Composition

[0131] Composition of the of the shake shake flask flask fermentation fermentation medium:10 medium: 10 g/L g/L glucose, glucose, 5.0 5.0 g/L g/L yeast yeast

extract extract powder, 10 g/L powder, 10 g/L rice rice bran, bran, 6.0 6.0 g/L g/L (NH 4)2SO (NH)SO, 4, 3.0 3.0 g/L g/L sodium sodium citrate, citrate, 2.02.0 g/Lg/L L- L-

glutamine, glutamine,1.0 1.0g/L L-serine, g/L 5.6 g/L L-serine, 5.6 K2HPO g/L 4, 3.0 KHPO, g/Lg/L 3.0 MgSO 4·7H MgSO4 2O,65 7HO, 65mg/L mg/LFeSO·7HO, FeSO4·7H2O, and 20 and 20 g/L g/L calcium calciumcarbonate, carbonate,and andpH=7.2. pH=7.2.

[0132] TakingEscherichia

[0132] Taking Escherichia coli coliBL21, BL21,HB101, HB101, JM109, JM109, DH10B, and MG1655 DH10B, and MG1655 asasthe theparent parent strain, strain, a a similar effect ofof1.3 similar effect 1.3toto1.5-fold 1.5-foldincrease increase in in tryptophan tryptophan production production can can also be also be achieved. achieved.

[0133] The

[0133] The foregoing foregoing description description only only illustratespreferred illustrates preferred embodiments embodiments of of thethe present present

disclosure, but the scope of the present disclosure is not limited thereto. Those skilled in the art disclosure, but the scope of the present disclosure is not limited thereto. Those skilled in the art

mayreadily may readily conceive conceiveofofvarious variousmodifications modificationsororsubstitutions substitutions within within the the technical technical scope scope

disclosed disclosed herein, herein, all allofofwhich whichshould should be be encompassed withinthe encompassed within theprotection protectionscope scopeofofthe the present present disclosure. Therefore,the disclosure. Therefore, theprotection protectionscope scopeofofthe the present present invention invention shall shall be be defined defined by by the the

claims. claims.

19

Claims (33)

1. WHAT IS CLAIMED WHAT IS CLAIMED IS: IS: 1. 1. An engineered An engineered strain, strain, wherein wherein the engineered the engineered strain strain is is obtained obtained by modification by modification from a parent from a parent

   strain, strain,and andthe theengineered engineered strain straincomprises comprises aagene gene encoding at least encoding at leastone oneof ofa apepD pepD mutant protein mutant protein

   or or aa fadR fadR mutant protein. mutant protein.
2. 2. The 2. The engineered strain of engineered strain of claim claim 1, 1, wherein wherein the the pepD mutantprotein pepD mutant proteinincludes includesatat least least one one of of

   mutations S21T, mutations S21T, G225A, G225A, and and A484K. A484K.
3. 3. The 3. The engineered strain of engineered strain of claim claim 1 1 or or claim claim 2, 2,wherein wherein the the pepD mutantprotein pepD mutant proteinincludes includes aa sequence havingatatleast sequence having least 80% sequenceidentity 80% sequence identitytotoan anamino aminoacid acidsequence sequence shown shown in SEQ in SEQ ID ID

   NO: 1. NO: 1.
4. 4. The engineered strain of claim 1, wherein the fadR mutant protein includes at least one of 4. The engineered strain of claim 1, wherein the fadR mutant protein includes at least one of

   mutations A140T mutations A140T and and L171I. L171I.
5. 5. 5. The The engineered strain of engineered strain of claim claim 4, 4, wherein wherein the the fadR fadR mutant protein includes mutant protein includes aa sequence having sequence having

   at at least least80% 80% sequence identity to sequence identity to an an amino acid sequence amino acid sequenceshown showninin SEQ SEQ ID ID NO: NO: 2. 2.
6. 6. The 6. The engineered strain of engineered strain of claim claim 1, 1, wherein wherein the the pepD mutantprotein pepD mutant proteinhas hasananamino aminoacid acid sequence shown sequence shown inin SEQ SEQ ID ID NO: NO: 1, and 1, and the the fadRfadR mutant mutant protein protein hasamino has an an amino acid sequence acid sequence

   shown in shown in SEQ ID NO: SEQ ID NO:2. 2.
7. 7. 7. The The engineered strain of engineered strain of claim claim 1, 1, wherein wherein the the pepD mutantprotein pepD mutant proteinincludes includesmutations mutationsS21T, S21T, G225A, andA484, G225A, and A484, andand thethe fadR fadR mutant mutant protein protein includes includes mutations mutations A140T A140T and L171I. and L171I.
8. 8. 8. The The engineered strain of engineered strain of claim claim 1, 1, wherein wherein the the modification modification includes includes enhancing anexpression enhancing an expression level of aa pepD level of pepD protein protein of the of the parent parent strain. strain.
9. 9. The 9. The engineered strain of engineered strain of claim claim 1, 1, wherein wherein the the modification modification includes includes weakening weakening ananexpression expression level of a FadR protein of the parent strain. level of a FadR protein of the parent strain.
10. 10. 10. The The engineered strain of engineered strain of any any one one of of claims claims 7-9, 7-9, wherein the modification wherein the includes knocking modification includes knocking out out a a fadR fadR gene in aa genome gene in genome ofofthe theengineered engineeredstrain strain including including the the pepD pepDmutant mutantprotein proteinwith with mutations S21T, mutations S21T,G225A, G225A,andand A484 A484 and and the fadR the fadR mutant mutant protein protein with with mutations mutations A140TA140T and and L171I, andreplacing L171I, and replacing aa promoter promoterofofaa gene geneencoding encodingthe thepepD pepD mutant mutant protein protein in in a genome a genome of the of the

    20 parent strain with a strong promoter. parent strain with a strong promoter.
11. 11. 11. The engineered The engineered strain strain of any of any oneclaims one of of claims 1-10, wherein 1-10, wherein the the parent parent strain strain isfrom is selected selected from any oneofofEscherichia any one Escherichia coli, coli, Corynebacterium Corynebacterium glutamicum, glutamicum, Bacillus Bacillus subtilis, or subtilis, or yeast cells. yeast cells.
12. 12. 12. The engineered The engineered strain strain of any of any one one of of claims claims 1-11, wherein 1-11, wherein the the parent parent strain strain isfrom is selected selected from one of aa strain one of strainIBEWQ, IBEWQ, a amutant mutantstrain strainIBEWQ-62, IBEWQ-62, a mutant a mutant strain strain IBEWQ-624, IBEWQ-624, an Escherichia an Escherichia

    coli Nissle1917, coli Nissle1917, Escherichia coli BL21, Escherichia coli Escherichiacoli BL21, Escherichia coliHB101, HB101, Escherichia Escherichia coli coli JM109, JM109,

    Escherichiacoli Escherichia coli DH10B, DH10B, oror Escherichia Escherichia coliMG1655. coli MG1655.
13. 13. 13. The The engineered strain of engineered strain of any any one one of of claims claims 1-12, 1-12, wherein underaa same wherein under sameculture culturecondition, condition, aa tryptophan production of the engineered strain is increased compared with the parent strain. tryptophan production of the engineered strain is increased compared with the parent strain.
14. 14. 14. A A method for constructing method for constructing an an engineered engineeredstrain strain with with aa high high tryptophan production, tryptophan production,

    comprising: comprising:

    obtaining the engineered obtaining the strain by engineered strain by modifying at least modifying at least one one of of aapepD protein or pepD protein or aa fadR fadR

    protein of a parent strain; wherein under a same culture condition, a tryptophan production of the protein of a parent strain; wherein under a same culture condition, a tryptophan production of the

    engineered strain engineered strain is is higher higher than than a tryptophan a tryptophan production production of thestrain. of the parent parent strain.
15. 15. 15. The The method ofclaim method of claim14, 14,wherein whereinthe themodification modificationincludes: includes: generating a pepD generating a mutantprotein pepD mutant proteinbybyintroducing introducinga amutation mutationatatananamino amino acidsite acid siteofofthe the pepD protein of the parent strain, wherein the mutation includes at least one of mutations S21T, pepD protein of the parent strain, wherein the mutation includes at least one of mutations S21T,

    G225A, and A484K; G225A, and A484K;oror overexpressingaa gene overexpressing geneencoding encodingthe thepepD pepD protein protein or or thepepD the pepD mutant mutant protein protein using using a high- a high-

    copy plasmidasasaa vector; copy plasmid vector; or or replacing aa promoter replacing of aa gene promoter of encodingthe gene encoding thepepD pepDprotein proteinororthe thepepD pepDmutant mutant protein protein in in a a genome genome ofofthe theparent parentstrain strain with a strong with a strong promoter; or promoter; or

    improving stability of improving stability of mRNA transcribedfrom mRNA transcribed from thethe gene gene encoding encoding the the pepD pepD protein protein or the or the

    pepDmutant pepD mutantprotein. protein.
16. 16. 16. The The method ofclaim method of claim15, 15,wherein whereinthe themodification modificationincludes: includes: the pepD the mutantprotein pepD mutant proteinincluding includinga asequence sequencehaving having atat least80% least 80% sequence sequence identity identity toto anan

    aminoacid amino acidsequence sequenceshown shownin in SEQSEQ ID NO: ID NO: 1. 1.
17. 17. 17. The The method ofclaim method of claim16, 16,wherein whereinthe themodification modificationincludes: includes:

    21 knocking out a gene encoding the fadR protein of the parent strain; or knocking out a gene encoding the fadR protein of the parent strain; or generating a fadR generating a mutantprotein fadR mutant proteinby byintroducing introducingaamutation mutationatatan anamino aminoacid acidsite site of of the the fadR protein of fadR protein of the the parent parent strain, strain,wherein whereinthe themutation mutation includes includesat atleast one least ofof one mutations A140T mutations A140T and L171I; and L171I;or or replacing aa promoter replacing of aa gene promoter of encodingthe gene encoding theFadR FadRprotein proteinororthe theFadR FadR mutant mutant protein protein in in a a genomeofofthe genome theparent parentstrain strain with a weak with a promoter;oror weak promoter; inhibiting translationefficiency inhibiting translation efficiency or or reducing reducing stability stability of mRNA of mRNA transcribed transcribed from the gene from the gene encoding the FadR encoding the FadRprotein proteinororthe theFadR FadRmutant mutant protein. protein.
18. 18. 18. The The method ofclaim method of claim17, 17,wherein whereinthe thefadR fadRmutant mutant proteinincludes protein includesa asequence sequence having having at at least least

    80% sequence 80% sequence identitytotoananamino identity aminoacid acidsequence sequence shown shown in SEQ in SEQ ID2. ID NO: NO: 2.
19. 19. 19. The The method ofclaim method of claim18, 18,wherein whereinthe thepepD pepD mutant mutant protein protein hashas an an amino amino acidacid sequence sequence

    shownininSEQ shown SEQID ID NO:NO: 1, and 1, and thethe fadR fadR mutant mutant protein protein has has an amino an amino acid acid sequence sequence shownshown in SEQin SEQ ID NO:2.2. ID NO:
20. 20. The 20. methodofofany The method anyone oneofofclaims claims14-19, 14-19,wherein wherein theobtaining the obtaining theengineered the engineered strainincludes: strain includes: culturing theparent culturing the parentstrain strainin inculture culture media media including including different different concentrations concentrations of tryptophan of tryptophan

    and measuring and measuringa abiomass biomassinineach eachculture culturemedium; medium; determining determining aa growth growthrate rate of of the the parent parent strain strainbased based on on the the biomass; biomass; and and

    obtaining the engineered obtaining the strain capable engineered strain capable of of increasing increasing aa tryptophan tryptophan production by fermenting production by fermenting a strain with a faster growth rate in a culture medium including high-concentration tryptophan. a strain with a faster growth rate in a culture medium including high-concentration tryptophan.
21. 21. The 21. methodofofclaim The method claim20, 20,wherein whereina aconcentration concentrationofofthe thehigh-concentration high-concentrationtryptophan tryptophan isisininaa

    range of 50 g/L to 70 g/L. range of 50 g/L to 70 g/L.
22. 22. A 22. A mutant protein, wherein mutant protein, the mutant wherein the mutantprotein protein includes includes aa sequence sequencehaving havingatatleast least 80% 80%

    sequence identity to sequence identity to an an amino acid sequence amino acid sequenceshown shownin in SEQ SEQ ID NO: ID NO: 1 or 1a or a sequence sequence having having at at least least 80% sequenceidentity 80% sequence identity to to an an amino aminoacid acidsequence sequenceshown shownin in SEQ SEQ ID NO: ID NO: 2. 2.
23. 23. The 23. mutantprotein The mutant protein of of claim claim 22, 22, wherein whereinthe the mutant mutantprotein proteinincludes includesaa pepD pepDmutant mutant protein, protein,

    whereinthe wherein the pepD pepDmutant mutant proteinincludes protein includesatatleast least one one of of mutations mutationsS21T, S21T,G225A, G225A,andand A484K. A484K.
24. 24. The 24. mutantprotein The mutant protein of of claim claim 23, 23, wherein whereinthe the mutant mutantprotein proteinincludes includesaa fadR fadRmutant mutantprotein, protein, whereinthe wherein the pepD pepDmutant mutant proteinincludes protein includesatatleast least one one of of mutations mutationsA140T A140Tandand L171I. L171I.

    22
25. 25. A 25. A DNA molecule, DNA molecule, comprising comprising a gene a gene encoding encoding the the mutant mutant protein protein of any of any one one of claims of claims 19-21. 19-21.
26. 26. A 26. A gene expressioncassette, gene expression cassette, comprising the mutant comprising the mutantprotein protein of of any any one oneof of claims claims 19-21 19-21ororaa gene encodingthe gene encoding themutant mutantprotein. protein.
27. 27. A 27. A recombinant vector, comprising recombinant vector, comprisingthe themutant mutantprotein proteinofofany anyone oneofofclaims claims19-21 19-21orora agene gene encoding the mutant encoding the mutantprotein. protein.
28. 28. A use of the engineered strain of claim 1 in increasing tryptophan production. 28. A use of the engineered strain of claim 1 in increasing tryptophan production.
29. 29. The 29. use of The use of claim 28, further claim 28, further comprising: comprising:

    fermenting andculturing fermenting and culturing the the engineered engineeredstrain strain to to obtain obtain tryptophan tryptophan with with increased increased

    production. production.
30. 30. 30. The use of The use of claim 28, wherein claim 28, the pepD wherein the pepDmutant mutantprotein proteinincluded includedininthe theengineered engineeredstrain strain includes includes any one of any one of the the following: following:

    (A1) (A1) aa protein protein having an amino having an aminoacid acidsequence sequenceshown shown in in SEQSEQ ID NO: ID NO: 1; or1; or

    (A2) (A2) aa protein protein derived derived from the amino from the aminoacid acidsequence sequenceshown shownin in SEQ SEQ ID NO: ID NO: 1 containing 1 containing

    one ormore one or more amino amino acid acid substitutions, substitutions, deletions, deletions, and/or and/or insertions, insertions, and possessing and possessing a same a same function function as as the the protein proteinhaving having the the amino amino acid acid sequence shownininSEQ sequence shown SEQID ID NO:NO: 1; or 1; or

    (A3) (A3) aa protein protein having an amino having an aminoacid acidsequence sequencewith withatatleast least 99%, 99%,95%, 95%, 90%, 90%, 85%, 85%, or 80% or 80%

    homologytotoany homology anyone oneofofthe theamino amino acidsequences acid sequences defined defined in in (A1) (A1) andand (A2), (A2), andand possessing possessing a a same functionas same function as the the proteins proteins defined defined in in (A1) (A1) and (A2); or and (A2); or (A4) (A4) aa fusion fusion protein protein obtained obtained by by linking linking aa tag tag to toN-terminus N-terminus and/or and/or C-terminus of any C-terminus of any one one of the proteins of the proteinsdefined definedin in (A1) (A1) to (A3). to (A3).
31. 31. 31. The use of The use of claim 28, wherein claim 28, the fadR wherein the fadRmutant mutantprotein proteinincluded includedininthe the engineered engineeredstrain strain includes includes any one of any one of the the following: following:

    (A1) (A1) aa protein protein having an amino having an aminoacid acidsequence sequenceshown shown in in SEQSEQ ID NO: ID NO: 2; or2; or

    (A2) (A2) aa protein protein derived derived from the amino from the aminoacid acidsequence sequenceshown shownin in SEQSEQ ID NO: ID NO: 2 containing 2 containing

    one ormore one or more amino amino acid acid substitutions, substitutions, deletions, deletions, and/or and/or insertions, insertions, and possessing and possessing a same a same function function as as the the protein proteinhaving having the the amino acid sequence amino acid shownininSEQ sequence shown SEQID ID NO:NO: 2; or 2; or

    (A3) (A3) aa protein protein having an amino having an aminoacid acidsequence sequencewith withatatleast least 99%, 99%,95%, 95%, 90%, 90%, 85%, 85%, or 80% or 80%

    homologytotoany homology anyone oneofofthe theamino amino acidsequences acid sequences defined defined in in (A1) (A1) andand (A2), (A2), andand possessing possessing a a

    23 same functionas same function as the the proteins proteins defined defined in in (A1) (A1) and (A2); or and (A2); or (A4) (A4) aa fusion fusion protein protein obtained obtained by by linking linking aa tag tag to toN-terminus N-terminus and/or and/or C-terminus of any C-terminus of any one one of the proteins defined in (A1) to (A3). of the proteins defined in (A1) to (A3).
32. 32. 32. The use of The use of claim 28, wherein claim 28, the gene wherein the gene encoding encodingthe thepepD pepD mutant mutant protein protein includes includes a DNA a DNA

    moleculehaving molecule havingatatleast least 99%, 95%,90%, 99%, 95%, 90%, 85%, 85%, or 80% or 80% homology homology to asequence to a DNA DNA sequence defined defined by aa gene by pepDS21T,G225A, encodingaapepD²¹T, gene encoding G225A,A484K A484K mutant protein. mutant protein.
33. 33. 33. The use of The use of claim 28, wherein claim 28, the gene wherein the gene encoding encodingthe thefadR fadRmutant mutant proteinincludes protein includesa aDNA DNA moleculehaving molecule havingatatleast least 99%, 95%,90%, 99%, 95%, 90%, 85%, 85%, or 80% or 80% homology homology to asequence to a DNA DNA sequence defined defined A140T, L171I by a gene encoding a fadR by a gene encoding a fadR A140T, L171I mutant protein. mutant protein.

    24